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Keywords:

  • autophagy;
  • exocytosis;
  • gene multiplication;
  • Parkinson’s disease;
  • proteasome;
  • protein aggregation

Abstract

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References

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 α-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 α-synuclein has been associated with the disease in both familial and sporadic forms of PD. Thus, maintenance of the intraneuronal steady state levels of α-synuclein below the critical concentration is a key challenge neuronal cells are facing. Expression of the α-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 α-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 α-synuclein has significant implications in the development of PD, and understanding the mechanism may disclose potential therapeutic targets for PD and other related diseases.

Abbreviations used
CMA

chaperone-mediated autophagy

LB

Lewy body

LN

Lewy neurite

PD

Parkinson’s disease

SNpc

substantia nigra par compacta

SNPs

single nucleotide polymorphisms

TH

tyrosine hydroxylase

UPS

ubiquitin-proteasome system

Parkinson’s disease (PD) is a major age-related neurodegenerative disorder, characterized primarily by motor symptoms, such as resting tremor, rigidity, and bradykinesia, but also accompanied by various non-motor symptoms (Dauer and Przedborski 2003). Pathologically, PD is characterized by the loss of dopaminergic neurons in the substantia nigra par compacta (SNpc) and two types of abnormal protein deposits, called Lewy bodies (LBs) and Lewy neurites (Forno 1996). Although the cause of the disease is still unknown, several mutations have been identified in familial cases of the disease (Farrer 2006). The first mutation was identified in 1997 in the gene encoding a neuronal protein, α-synuclein. Since then, two more autosomal dominant missense mutations and several gene multiplication mutations in this gene have been identified in familial PD. The link between α-synuclein and PD, including idiopathic PD, was solidified by the finding that fibrillar aggregates of this protein are components of LBs and Lewy neurites (Spillantini et al. 1998). Biochemical studies showed that α-synuclein forms amyloid fibrils that are structurally related to the ones found in LBs (Conway et al. 2000). Furthermore, all the missense mutations in α-synuclein that are linked with familial PD accelerated the aggregation of the protein (Cookson 2005). The importance of α-synuclein aggregation in neurodegeneration was further supported by animal model studies, in which over-expression of wild type and mutant forms of α-synuclein led to neuronal loss and LB-like inclusion formation (Maries et al. 2003).

In light of the occurrence of LBs and glial α-synuclein depositions in other neurological diseases, including dementia with LBs, Alzheimer’s disease, and multiple system atrophy (Jellinger 2003), the role of α-synuclein aggregation may not be limited to PD, but may extend to a variety of brain disorders. We are now beginning to understand the biochemical and cell biological mechanisms of α-synuclein aggregation. One of the critical factors controlling the aggregation process is the concentration of protein. In the case of PD, evidence has been accumulating that underlines the importance of elevated α-synuclein concentration. In this review, we will provide (i) genetic and pathological evidence for the importance of intraneuronal α-synuclein concentration in PD, (ii) a biophysical account of why the concentration of this protein is important, and (iii) the current state of knowledge about the mechanisms of controlling the steady state levels of α-synuclein, covering the literature on biosynthesis, breakdown, and secretion of the protein.

Structure and function of α-synuclein

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References

α-Synuclein is a member of the synuclein family which includes β-synuclein and γ-synuclein (George 2001). Human α-synuclein is composed of 140 amino acids that contain highly conserved amphipathic N-terminal regions, with seven repeats of the KTKEGV consensus motif and highly variable acidic C-terminal regions (Cookson 2005). α-Synuclein also contains a central hydrophobic non-amyloid-β component domain, through which amyloidogenic intermolecular interaction might occur (Giasson et al. 2001). In isolation, α-synuclein lacks a stable structure, and is hence classified as a natively unfolded protein (Weinreb et al. 1996; Bussell and Eliezer 2001; Eliezer et al. 2001; Uversky et al. 2001a, 2002). However, numerous transient structures constitute a ‘conformational ensemble,’ which often involves many conformational species with long-range interactions between the N- and C-terminal regions (Bertoncini et al. 2005; Dedmon et al. 2005; Lee et al. 2005b; Sandal et al. 2008). This structural plasticity of α-synuclein may confer the ability to adopt various conformations depending on the environment, prompting the term ‘chameleon’ (Uversky 2003). The same property might also allow the pathological transition from monomers to oligomers and filamentous forms. The structural properties and its implications in aggregation and pathogenesis were reviewed extensively elsewhere (Dev et al. 2003; Fink 2006; Uversky 2007). Although the conformation of α-synuclein has been characterized extensively in vitro with a battery of biophysical methods, little is known about the native structure of this protein in living cells, which may not be the same as those observed in vitro.

The biological function of α-synuclein is not clear, but its abundance in pre-synaptic nerve terminals and its ability to bind to lipid membranes indicate its function in neural transmission (Iwai et al. 1995; Davidson et al. 1998). α-Synuclein knockout studies showed significant changes in neurotransmission, synaptic vesicle recycling, and number of synaptic vesicles (Abeliovich et al. 2000; Cabin et al. 2002; Liu et al. 2004; Yavich et al. 2004). Knockout studies also suggested its role in fatty acid metabolism (Castagnet et al. 2005; Golovko et al. 2005). Another set of studies suggested that α-synuclein is a molecular chaperone. In support of this suggestion, it was demonstrated that α-synuclein prevented the aggregation of thermally and chemically denatured proteins (Kim et al. 2000; Souza et al. 2000), was up-regulated by stress-inducing reagents (Vila et al. 2000; Gomez-Santos et al. 2002; Manning-Bog et al. 2002; Kalivendi et al. 2004), and protected cells from cytotoxic insults (Manning-Bog et al. 2003; Albani et al. 2004).

Concentration dependence of protein aggregation

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References

Protein aggregates that are associated with neurodegenerative diseases generally have highly ordered polymeric structures formed by an alternative, off-pathway folding process, which competes with the on-pathway native folding process. This off-pathway folding involves self-assembly of partially folded folding intermediates that accumulate due either to folding defects in newly synthesized polypeptides or damage to pre-existing proteins (Horwich 2002). Although α-synuclein belongs to a group of ‘natively unfolded’ proteins, amyloidogenic aggregation of this protein also requires partially folded intermediates (Uversky et al. 2001a). This partially folded form is unstable and the folding is readily reversible, but concentration of proteins in this conformation leads to dimerization of the protein, which in turn stabilizes the conformation (Uversky et al. 2001c). Therefore, at least in vitro, stabilization and accumulation of partially folded intermediates are critical initial steps of α-synuclein aggregation. Interestingly, aggregation of folding intermediates is sequence-specific, allowing only homotypic associations (London et al. 1974). Recently, it was demonstrated that protein aggregation occurs through highly specific self-association in living cells that co-express two unrelated misfolded proteins (Rajan et al. 2001). This sequence specificity in the assembly process provides the theoretical basis for the concentration dependence of protein aggregation. Specifically, the concentration of partially folded intermediates is the critical factor controlling the aggregation process. Concentration of the intermediate is under the control of various factors including oxidative stress, activities of protein degradation systems, and chaperone activities (Fig. 1). The levels of the intermediate are also in equilibrium with the other forms of the polypeptide, including the unfolded polypeptides and the native protein (in case of α-synuclein, the native form in the cytoplasm is not clearly understood). Therefore, unlike the native folding process, which is independent of protein concentration, protein aggregation is highly sensitive to the steady state levels of the protein, whether or not the protein is natively folded.

image

Figure 1.  Steady state level of α-synuclein and its aggregation. Protein aggregation involves the self-assembly of folding intermediates, and is therefore a concentration-dependent process. As the folding intermediates are in equilibrium with the newly synthesized unfolded polypeptides, as well as with the native protein, an increase in the steady state level of protein promotes protein aggregation. The level of α-synuclein is affected at both the synthesis and breakdown levels by various genetic and environmental factors that cause the development of PD.

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Evidence supporting the importance of α-synuclein steady state levels in PD

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References

Genetics

Gene multiplication

Some studies have demonstrated that the steady state levels of α-synuclein are affected by genetic variations, such as gene multiplications and polymorphisms. Both duplication and triplication of the genomic locus containing the α-synuclein gene are responsible for some cases of rare familial PD (Singleton et al. 2003; Chartier-Harlin et al. 2004; Ibanez et al. 2004; Ross et al. 2008). The genomic triplication has been reported in Iowan and Swedish-American families. Triplication of the allele resulted in twofold increased gene dosage, which was confirmed by fluorescent in situ hybridization in the Iowan family (Singleton et al. 2003). Both α-synuclein mRNA and protein levels were nearly doubled, and α-synuclein proteins formed sodium dodecyl sulfate-insoluble aggregates in the brain samples of the patients (Miller et al. 2004). The Swedish-American patients had another type of α-synuclein triplication, and also showed an increase in mRNA and protein levels (Farrer et al. 2004). Clinically, affected members of both families show typical PD symptoms, including l-DOPA responsive parkinsonism, postural hypotension, and intellectual impairment (Farrer et al. 2004). However, these cases show an earlier onset of disease and more rapid disease progression than sporadic cases. Pathologically, both family members had widespread LBs and serious neuronal degeneration in the SNpc and the locus ceruleus (Muenter et al. 1998; Farrer et al. 2004). Additionally, duplication of the α-synuclein genomic locus has been observed in familial PD patients, such as in French and Italian families. These patients have three copies of the α-synuclein gene instead of two (Chartier-Harlin et al. 2004; Ibanez et al. 2004). The clinical features of these duplication cases are indistinguishable from those of idiopathic PD (Chartier-Harlin et al. 2004).

The ages of onset in these multiplication cases show a strong gene dosage effect, providing compelling evidence for the causal role of high α-synuclein burden in PD. Patients with heterozygous duplication mutations typically develop PD in their fifties, while the triplication mutations with four copies of α-synuclein cause PD in patients in their thirties (Chartier-Harlin et al. 2004). Among the duplication cases, one case of homozygous mutation was found, and this patient showed an earlier age of onset and death than the heterozygous patients (Ikeuchi et al. 2008). Furthermore, the increased α-synuclein gene dosage causes shorter disease duration and more severe Lewy pathology (Ikeuchi et al. 2008). Interestingly, duplication of α-synuclein has also been identified in non-familial PD patients; from 1106 Korean patients exhibiting parkinsonism, two patients who had no familial histories were found to have α-synuclein gene duplications (Ahn et al. 2008). Thus, although these multiplication mutations are not frequently observed in PD patients, these studies suggest that the steady state level of α-synuclein is a critical determinant for both the development and progression of PD.

Polymorphism

Polymorphic dinucleotide repeat sequences on the α-synuclein promoter (Rep1 microsatellite) were first reported in 1996 (Xia et al. 1996) and were associated with risk of PD (Kruger et al. 1999; Tan et al. 2000, 2003; Farrer et al. 2001; Pals et al. 2004; Mellick et al. 2005; Hadjigeorgiou et al. 2006). Rep1 consists of (TC)x(TT)(TC)y(TA)z(CA)wrepeated sequences, where x, y, z and w indicate repeat numbers, and is located ∼10 kb upstream of the gene. Association studies reported that increased length of Rep1 was associated with an earlier disease onset, by about 5 years (Kay et al. 2008) and this led to increased mRNA levels of α-synuclein (Chiba-Falek et al. 2003). Reporter analyses in 293T and SH-SY5Y cells showed that Rep1 could act as a transcription modulator (Chiba-Falek and Nussbaum 2001). Although some studies with small sample sizes found no association (Parsian et al. 1998; Khan et al. 2001; Spadafora et al. 2003) or reverse associations (Izumi et al. 2001; Mizuta et al. 2002), a large-scale study has recently confirmed the association (Maraganore et al. 2006).

Association studies also identified several single nucleotide polymorphisms (SNPs) associated with PD in or near the α-synuclein gene. Multiple case-control studies have revealed SNPs at the 3′-region of the α-synuclein gene that were strongly associated with incidence of PD and increased α-synuclein expression in German (Mueller et al. 2005), Japanese (Mizuta et al. 2006), and Norwegian (Myhre et al. 2008) PD patients. Two polymorphic bases in the promoter region of the gene (−116C > G and −668T > C) were also associated with the risk of PD in German patients (Holzmann et al. 2003). A reporter gene assay showed that the haplotype with high PD risk (−668C/−116G) has higher transcriptional activity than others. Another PD-associated polymorphism, RasI T-to-C substitution, is also located in the region upstream of the gene (111 bp downstream of Rep1) and affects the level of α-synuclein mRNA in PD patients (Wang et al. 2006). The Northern Central and Southeastern European case study confirmed that both the 5′-region and 3′-region SNPs mentioned above are associated with PD susceptibility (Winkler et al. 2007). Taken together, both genetic linkage analyses and association studies support the hypothesis that increased α-synuclein expression is a determining factor in PD pathogenesis.

Postmortem studies

Analyses of postmortem brain samples of sporadic PD patients showed changes in α-synuclein expression levels. Several studies have shown that α-synuclein mRNA levels were significantly increased in either SNpc or midbrain samples of PD patients compared with the control group, whereas the variations in frontal cortex samples were not significant (Solano et al. 2000; Rockenstein et al. 2001; Chiba-Falek et al. 2006). In contrast, some studies have reported a decrease (Neystat et al. 1999; Beyer et al. 2004; Kingsbury et al. 2004; Dachsel et al. 2007) or no alteration (Tan et al. 2005) of α-synuclein mRNA levels in the SNpc of PD patients. Recently, Grundemann et al. (2008) investigated this problem using the UV-laser-microdissection method and reported significant increases in α-synuclein mRNA levels, as well as tyrosine hydroxylase and neuron-specific enolase mRNA levels, in individual neuromelanin-positive SNpc neurons of sporadic PD patients. On the other hand, accurate measurements of protein levels in single neurons from brain tissue are difficult to achieve. However, biochemical analyses observed significantly increased high-molecular weight α-synuclein protein aggregates in the SNpc and other brain regions of PD patients (Baba et al. 1998; Campbell et al. 2001; Tofaris et al. 2003). Overall, the level of α-synuclein is elevated in sporadic PD, and thus, lowering the α-synuclein burden seems to be a key challenge facing neurons under disease-developing conditions.

Aging is the best-established risk factor for PD, and α-synuclein protein levels are significantly elevated in aged human SNpc (Chu and Kordower 2007). There is a leap in expression at birth, and later in life, protein levels of α-synuclein are increased again in human cortex and hippocampus (Bayer et al. 1999). Interestingly, human adult samples have increased amounts of α-synuclein protein compared with fetal samples, but without significant elevation of α-synuclein mRNA (Bayer et al. 1999), suggesting the regulation of translation and/or degradation. Aging-associated increases in α-synuclein expression have also been observed in both human and non-human primates (Chu and Kordower 2007). This study has shown that accumulation of soluble (proteinase K sensitive) α-synuclein in the cell bodies of SNpc neurons preceded the formation of proteinase K-resistant inclusion bodies, and the soluble α-synuclein accumulation was associated with a reduction of the dopaminergic phenotype [tyrosine hydroxylase (TH)-immunoreactivity]. The immunohistochemistry results of this study were supported by the quantitative western analysis showing that a group of patients over 80-year old had higher amounts of α-synuclein protein than a group 49- to 58-year old (Li et al. 2004b).

Toxicology

There is a large body of evidence demonstrating that exposure to specific environmental agents may cause PD (Marion 2001). Administration of these agents, such as MPTP, rotenone, and paraquat to animals caused dopaminergic degeneration and PD-like behavioral abnormalities (Burns et al. 1983; Betarbet et al. 2000; Manning-Bog et al. 2002). The mechanism underlying the interaction in the pathogenesis between these environmental agents and genetic factors has been one of the most critical questions in PD research. Animal model studies have shown an association between the quantitative increase in α-synuclein and the toxicant-induced PD-like phenotypes. MPTP is the most widely studied and the most reliable PD-inducing agent in both humans and animals. In the brain, it is converted to MPP+ by monoamine oxidase B in astrocytes (Brooks et al. 1989), which may then selectively enter into dopaminergic neurons through the dopamine transporter, inhibiting mitochondrial complex I, and causing selective degeneration of dopaminergic neurons (Javitch et al. 1985). Chronic MPTP-administration caused the elevation of mRNA and/or protein levels of α-synuclein in TH-positive SNpc neurons in mice and various non-human primate species (Vila et al. 2000; Kuhn et al. 2003). Tissue culture studies have also shown that MPP+ treatment increased the expression and aggregation of α-synuclein (Gomez-Santos et al. 2002; Kalivendi et al. 2004). In addition, 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), an endogenous neurotoxin that causes parkinsonism in rodents and non-human primates, also increased the expression of α-synuclein in SH-SY5Y cells (Shavali et al. 2004).

Recently, several studies with α-synuclein null mice have suggested that this protein is an important mediator of MPTP-induced dopaminergic degeneration (Dauer et al. 2002; Drolet et al. 2004; Fornai et al. 2005). These studies collectively showed that deletion of the α-synuclein gene conferred a significant degree of resistance to MPTP in TH-positive SNpc neurons. Taken together, these studies indicate that α-synuclein plays a role in dopaminergic degeneration induced by exposure to environmental toxicants, and thus, the level of this protein might be a modulatory factor through which the extent of damage caused by the toxicants is determined.

Regulation of α-synuclein levels

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References

Synthesis

α-Synuclein is expressed predominantly in the CNS, especially in the hippocampus, olfactory bulb, striatum, and cerebellum, but it is also expressed at low levels in other tissues (Ueda et al. 1993; Iwai et al. 1995). At the subcellular level, α-synuclein is enriched in the pre-synaptic nerve terminals (Iwai et al. 1995). Expression of α-synuclein is regulated during development. It has been shown that α-synuclein levels increase throughout embryonic development in mice (Hsu et al. 1998) and rats (Petersen et al. 1999) and that the timing of α-synuclein expression varies in discrete neuronal populations during human development (Raghavan et al. 2004). In hippocampal culture, translocation of α-synuclein from the soma to the synaptic terminals occurs in later stages of neuronal development (Withers et al. 1997). These results suggest that both the expression and subcellular localization of α-synuclein is temporally regulated.

During adult life, several factors affect α-synuclein expression. As discussed above, toxins that are known to cause PD also increase expression of α-synuclein. Expression of α-synuclein is also regulated by reagents other than PD-related toxins. Valproic acid, a mood-stabilizer, has been shown to increase α-synuclein mRNA levels through histone deacetylase inhibition (Leng and Chuang 2006). Overuse of cocaine has also been shown to elevate mRNA levels of α-synuclein in the human substantia nigra and striatum (Mash et al. 2003; Qin et al. 2005). Furthermore, α-synuclein mRNA levels were elevated in patients with alcoholism (Bonsch et al. 2004). Importantly, it was also shown that the levels of α-synuclein-immunoreactive neurons were higher in aged human and monkey SNpc than in young specimens (Chu and Kordower 2007), suggesting that α-synuclein expression is regulated by aging, the most prominent risk factor for PD and other neurodegenerative diseases. Despite the increase in the protein levels, α-synuclein mRNA levels decreased with aging in human brain (Li et al. 2004b).

Regulation of α-synuclein expression has been investigated at the transcription level. The promoter of α-synuclein is TATA-less (Liang and Carr 2006) and is regulated by nerve growth factor and basic fibroblast growth factor through the mitogen-activated protein kinase pathway (Clough and Stefanis 2007). Several transcription factors have been identified as regulators of α-synuclein gene expression. CCAAT/enhancer-binding protein β regulates the α-synuclein gene through binding of the CCAAT motif (Gomez-Santos et al. 2005). Nurr-1, an orphan nuclear receptor has also been shown to regulate the expression of α-synuclein (Yang and Latchman 2008). On the other hand, poly-(ADP-ribose) polymerase-1 acts as a negative regulator through its binding to the Rep1 microsatellite (Chiba-Falek et al. 2005). Additionally, a bioinformatics search of transcription factor binding sites in the human, rat, and mouse genes predicted that several general transcription factors, such as SP1, AP-1, AP-4, GATA-1, and YY1, bind to the α-synuclein promoter (Liang and Carr 2006). Studies on the role of these transcription factors in α-synuclein expression will enhance our understanding of the mechanism underlying the gene expression changes, both increase and decrease, caused by the above-mentioned neurotoxins and drugs as well as by aging.

Degradation

α-Synuclein has a relatively long half-life, which increases with age in neurons (Li et al. 2004b). This indicates that its breakdown is not highly dynamic under normal circumstances. However, with protein breakdown being one of the main mechanisms for controlling steady state levels of a protein, the mechanism of α-synuclein degradation has been extensively investigated. Despite the diverse set of experiments reported over the past several years, it appears that the jury is still out concerning the major mechanism of α-synuclein degradation in neurons.

Ubiquitin-proteasome system

A wealth of data suggested a critical role played by ubiquitin-proteasome system (UPS) defects in the pathogenesis of PD (McNaught et al. 2001), leading to studies aimed at determining the role of the UPS in α-synuclein metabolism. Selective chemical inhibitors of proteasomal enzyme activities, such as β-lactone and epoxomicin, blocked the degradation of α-synuclein in tissue culture (Bennett et al. 1999; Webb et al. 2003). Furthermore, proteasomal inhibition, either by chemical inhibitors or by amino acid substitution in a proteasomal subunit, led to the accumulation of α-synuclein and LB-like inclusion body formation (Rideout et al. 2001; McNaught et al. 2002; Hyun et al. 2003; Li et al. 2004c). Although α-synuclein can be ubiquitinated at multiple sites in an in vitro reaction (Liu et al. 2002) and some α-synuclein is ubiquitinated in the brains of patients with LB diseases (Hasegawa et al. 2002), ubiquitination does not appear to be essential for proteasomal degradation of α-synuclein (Tofaris et al. 2001; Liu et al. 2003).

Interestingly, α-synuclein binds to specific subunits of the proteasome complex, such as S6′ (Snyder et al. 2003) and Tat binding protein 1 (Ghee et al. 2000) of the 19S regulatory complex. The aggregates seem to have high affinity for the 20S catalytic complex (Lindersson et al. 2004), and show strong inhibition of the enzyme activity. When over-expressed in cells, PD-linked mutant forms of α-synuclein reduced cellular proteasomal degradation, while wild type protein showed little effect (Stefanis et al. 2001; Tanaka et al. 2001; Petrucelli et al. 2002). In a recent study, Zhang et al. (2008) showed that α-synuclein protofibrils, but not the monomer or dimer, inhibited both ubiquitin-independent degradation of unstructured protein substrates and ubiquitin-dependent degradation of folded proteins. If α-synuclein is indeed a substrate of the proteasome, α-synuclein-mediated proteasomal impairment could create a vicious cycle leading to α-synuclein accumulation and aggregation. However, the role of proteasome in α-synuclein breakdown has been questioned in other studies and needs further clarification (Ancolio et al. 2000; Biasini et al. 2004; Vogiatzi et al. 2008).

Autophagy

Autophagy refers to a protein degradation mechanism mediated by the lysosome (Klionsky 2007). Depending on how substrates are delivered to the lysosome, three distinct pathways for autophagy have been identified: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (Mizushima et al. 2008). Interestingly, the activities of these autophagy pathways are decreased with aging, implicating autophagy substrate accumulation in the disease process (Cuervo et al. 2005). It has been reported that deletion in neurons of atg5 or atg7, essential genes in autophagosome formation, caused the accumulation of poly-ubiquitinated proteins and subsequent neurodegeneration in mice (Hara et al. 2006; Komatsu et al. 2006). This suggests that autophagy dysfunction may be responsible for the development of neurodegenerative diseases. One extreme example of such autophagic dysfunction is lysosome storage diseases, which are inherited metabolic disorders caused by deficiency of lysosomal enzymes, leading to abnormal accumulation of autophagy substrates and neurodegeneration (Kiselyov et al. 2007). In addition, increased autophagic organelles are one of the common pathological characteristics in chronic neurodegenerative diseases, including PD (Rubinsztein et al. 2005; Pan et al. 2008).

The role of autophagy in the degradation of α-synuclein has been addressed in several studies. Activation of macroautophagy increased α-synuclein clearance and localization of α-synuclein in acidic organelles in PC12 cells (Webb et al. 2003). Inhibition of lysosomal function by deacidifying agents also caused the accumulation of intracellular α-synuclein (Webb et al. 2003; Lee et al. 2004). In addition, α-synuclein has been shown to be directly translocated to and degraded by the lysosome via CMA (Cuervo et al. 2004; Vogiatzi et al. 2008). Interestingly, mutant forms of α-synuclein strongly bound to Lamp2a, the receptor for CMA substrate proteins, without subsequent translocation into the lysosome, thereby physically blocking degradation of other CMA substrates, including wild type α-synuclein (Cuervo et al. 2004). Furthermore, dopamine modification of α-synuclein impaired the degradation of this protein by CMA, and the modified α-synuclein blocked the degradation of other CMA substrates, possibly explaining the selective degeneration of dopaminergic neurons in PD (Martinez-Vicente et al. 2008).

In accordance with the ability of autophagy to clear aggregation-prone proteins, several studies showed that enhancement of autophagy with small molecules may be a potential therapeutic strategy for neurodegenerative diseases including PD. Activation of autophagy with rapamycin, a inhibitor of the mammalian target of rapamycin, protected neurons in fly and mouse models of Huntington’s disease (Ravikumar et al. 2004). In a recent study, a screen for small molecule modulators of autophagy yielded several small molecule enhancers of autophagic clearance of mutant huntingtin fragments and A53T α-synuclein (Sarkar et al. 2007b). These enhancers attenuated mutant huntingtin-induced neurodegeneration in cell and fly models. In another study, lithium, an inhibitor of inositol monophosphatase, was shown to increase the clearance of mutant huntingtin and the PD-linked α-synuclein mutants by inducing autophagy (Sarkar et al. 2005). A disaccharide trehalose is yet another small molecule that enhances the clearance of mutant huntingtin and mutant α-synuclein through the induction of autophagy (Sarkar et al. 2007a). These studies provide evidence in favor of the protective function of autophagy against neurodegeneration.

However, autophagy may also function in programmed cell death through self-digestion of injured cells (Bursch 2001). Histological observations of the increase in autophagic signs in neurodegenerative diseases confuse, rather than settle, the question of whether autophagy is a part of the neuronal death process or a protective mechanism against it. Although this issue needs to be addressed in future studies, it has been suggested that autophagy is induced to protect against early stage neurodegeneration (Boland and Nixon 2006; Bandhyopadhyay and Cuervo 2007) but that over-activation of autophagy in neurons eventually leads to neuronal death (Takacs-Vellai et al. 2006). Therefore, despite the promise seen in animal models, therapeutic applications to neurodegenerative diseases of prolonged autophagy activation should be approached with caution.

What is the main mechanism of α-synuclein breakdown?

Despite the increasing number of publications on the issue, the mechanism related to the degradation of normal α-synuclein remains controversial. In purified systems, both proteasomes and lysosomes are capable of degrading α-synuclein (Liu et al. 2003; Cuervo et al. 2004). In tissue culture and animal models, α-synuclein breakdown was influenced by small molecule modulators of UPS or autophagy (see above). The critical question that remains is how α-synuclein degradation pathways decide between the UPS and autophagy. One report suggested that the co-chaperone carboxyl terminus of heat-shock protein 70-interacting protein acts as a molecular switch involved in the decision between the UPS and autophagy pathways (Shin et al. 2005). Another important determinant may be the assembly state of α-synuclein. As the proteasome degrades substrates by passing the unfolded polypeptides through the narrow catalytic cavity, oligomeric assemblies are sterically incompatible as proteasome substrates. Consistent with this, there has been a report showing that oligomeric forms of α-synuclein were cleared by autophagy, not by the UPS (Lee et al. 2004). While this issue remains unresolved, we speculate that the decision between the UPS and autophagy in α-synuclein breakdown likely depends on the specific physiological state of the cell and the stage of the pathogenic process, as well as on the physical state of the substrate protein. Additionally, future studies should also address the role of other proteases, including matrix metalloproteinases, neurosin, and calpain-1, which have been shown to cleave normal or aggregated forms of α-synuclein in vitro (Iwata et al. 2003; Mishizen-Eberz et al. 2003; Sung et al. 2005).

Exocytosis of α-synuclein

Results from recent studies by our group and others suggest that exocytosis of α-synuclein into the extracellular space is a mechanism for controlling the intracellular level of this protein. Despite the fact that α-synuclein is typically a cytosolic protein, secretion of this protein from cells has been demonstrated in several tissue culture systems, including primary neurons (El-Agnaf et al. 2003; Lee et al. 2005; Sung et al. 2005). Although the mechanism is not well understood, vesicle-mediated exocytosis appears to be the primary mechanism responsible for the secretion of α-synuclein, which must involve the translocation of the protein across the vesicular membrane (Lee et al. 2005a). Electron microscopy and density gradient ultracentrifugation experiments have suggested that the vesicles containing α-synuclein have morphologies and sedimentation properties similar to the dense core vesicles (Lee et al. 2005a), but their exact identities remain unknown.

Only a small amount of cellular α-synuclein is translocated into the vesicles and secreted, and therefore, vesicle entry must be a selective process. Our recent study suggests that misfolded/damaged proteins are selected and translocated into vesicles, and subsequently discarded from cells by exocytosis (Lee et al., manuscript submitted). This predicts the accumulation of misfolded α-synuclein in specific vesicle populations, leading to aggregation. In fact, α-synuclein aggregation preferentially occurred in vesicles, and these aggregates were exocytosed from cells (Lee et al. 2005a). Although little is known about the physiological and pathological function of extracellular α-synuclein, its potential role in the initiation and progression of PD has been discussed elsewhere (Lee 2008).

The presence of extracellular α-synuclein has been demonstrated in human CSF and blood plasma samples from both PD and normal subjects (Borghi et al. 2000; El-Agnaf et al. 2003). Although it is controversial whether the level of extracellular α-synuclein changes in disease (Borghi et al. 2000; El-Agnaf et al. 2003; Lee et al. 2006; Tokuda et al. 2006), measurement using a novel ELISA specific for the assembled forms of α-synuclein showed significantly elevated levels of oligomeric α-synuclein in blood plasma and postmortem CSF from PD patients (El-Agnaf et al. 2006).

How is the extracellular α-synuclein cleared? One mechanism for clearance relies on degradation by extracellular proteolytic enzymes. It has been shown that matrix metalloproteases may cleave both recombinant α-synuclein and α-synuclein secreted from human neuroblastoma cells (Sung et al. 2005). However, the proteolytic fragments may facilitate aggregation of α-synuclein and the resulting aggregates show higher toxicity (Sung et al. 2005). Thus, it is not clear whether the matrix metalloprotease-mediated cleavage of extracellular α-synuclein is a mechanism for disposal of the protein, or whether it enhances the debilitating effects of the protein by producing more harmful species. Another mechanism for clearing extracellular α-synuclein involves uptake of the protein by neighboring cells (Sung et al. 2001; Zhang et al. 2005; Ahn et al. 2006; Lee et al. 2008b). In particular, aggregated forms of α-synuclein are taken up by cells via receptor-mediated endocytosis, followed by endosomal trafficking and lysosome-mediated breakdown (Lee et al. 2008b). Breakdown of extracellular α-synuclein aggregates via the endo-lysosomal pathway occurs in all the major cell types in brain parenchyma, such as neurons, astrocytes, and microglia, of which microglia showed the most efficient uptake and clearance of the aggregates (Lee et al. 2008a). In summary, misfolded intracellular α-synuclein may be cleared from the cytoplasm by selective translocation into secretory vesicles and subsequent exocytosis. The secreted extracellular α-synuclein, especially the aggregated forms, are then taken up by various types of adjacent cells, and degraded by the lysosomal system.

Subcellular localization

α-Synuclein is predominantly a neuronal protein and it is abundant in pre-synaptic terminals (Iwai et al. 1995). After biosynthesis in neuronal cell bodies, α-synuclein is transported along the axons mostly in the slow component a/b, while about 15% is transported in the fast component, possibly through interaction with vesicular membranes (Jensen et al. 1998). The extent to which the fast component is involved in the axonal transport of α-synuclein is controversial (Li et al. 2004a). Whatever the mechanism of the transport, impaired axonal transport would contribute to abnormal accumulation of α-synuclein in perikarya and axons. PD-linked mutations may alter axonal transport of α-synuclein. A30P mutation of α-synuclein blocked binding of α-synuclein to vesicles and axonal transport (Jensen et al. 1998), thereby inducing α-synuclein accumulation in the cell body in both cultured neurons and transgenic mice (Kahle et al. 2000; Saha et al. 2004). Furthermore, axonal transport of α-synuclein slows significantly with aging (Li et al. 2004a).

Abnormal accumulation of α-synuclein in specific cellular organelles may increase local concentration of this protein even without altering the total level. For example, nuclear localization of α-synuclein was promoted by treatment with paraquat (Goers et al. 2003). Amino acids 134–139 of α-synuclein seem to be important for this localization (Yu et al. 2007). Additionally, exposure of cells to various stresses led to translocation of α-synuclein to the mitochondria (Cole et al. 2008; Parihar et al. 2008; Shavali et al. 2008).

Concluding remarks

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References

A vast amount of evidence from genetic, pathological, and toxicological studies points to the importance of increased steady state levels of α-synuclein in disease development. Furthermore, the fact that α-synuclein expression is associated with pro-survival effects, in particular in the setting of synaptic dysfunction (Chandra et al. 2005), suggests that maintaining the proper amount of expression is important for neuronal survival. Cellular α-synuclein concentration is under the control of transcriptional regulation and degradation by the UPS and autophagy (Fig. 2). Some of the cytoplasmic α-synuclein is translocated into vesicles and secreted from cells, presumably lowering the cytoplasmic concentration of the protein. Furthermore, abnormal localization of α-synuclein in particular intracellular organelles may increase the local concentration of this protein. The steady state levels of α-synuclein are likely regulated by the interplay between these mechanisms. There are several outstanding questions related to these mechanisms. What are the signaling mechanisms linking exposure to environmental or endogenous toxicants to α-synuclein gene induction? What are the major breakdown mechanisms for α-synuclein in the normal and disease states? What is the mechanism of α-synuclein exocytosis, and how does it contribute to α-synuclein quality control? How do the different mechanisms cross-talk to maintain the appropriate levels of α-synuclein?

image

Figure 2.  Multiple points of impaired regulation of neuronal α-synuclein concentration. Genetic defects and environmental agents cause deregulated transcription of the α-synuclein gene elevating the cytoplasmic concentration of the protein. Impaired degradation or axonal transport may cause cytoplasmic and axonal accumulation of the protein. Exocytosis may be another mechanism for the removal of damaged α-synuclein, impairment of which would lead to the deposition of the protein. Abnormal localization elevates the local concentration of the protein without affecting the total level of the protein.

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Although our discussion in this review is focused on the regulation of steady state levels of total cellular α-synuclein, perhaps the most relevant parameter is the concentration of highly aggregation-prone conformer(s) of this protein. In contrast to test tube conditions where conformers of a single polypeptide are in simple equilibrium, cellular proteins are situated in a more dynamic environment where molecular interactions, covalent modifications of side chains, and enzyme-mediated peptide hydrolysis prevail. Some of these cytoplasmic events, or alterations in these events, are known to promote cellular α-synuclein oligomerization/aggregation, perhaps by favoring the aggregation intermediate conformation. These include interaction with lipids (Perrin et al. 2001; Cole et al. 2002; Lee et al. 2002), interaction with metal ions (Uversky et al. 2001b), interaction with other proteins (Engelender et al. 1999; Alim et al. 2002), phosphorylation (Fujiwara et al. 2002), oxidation and nitration (Norris et al. 2003), and proteolytic truncation of the C-terminus (Li et al. 2005; Liu et al. 2005). Therefore, the conformational diversity of α-synuclein and the dynamic shifts between conformers, especially in the cytoplasmic context, is an important issue. Solving this problem along with the development of tools that lower α-synuclein levels holds promise for advancing the state of PD therapeutics.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References

This work was supported by a grant from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology (M103KV010021-06K2201-02110), by the Diseases Network Research Program of the Ministry of Education, Science and Technology (2007-04303), and by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (R01-2007-000-20200-0).

References

  1. Top of page
  2. Abstract
  3. Structure and function of α-synuclein
  4. Concentration dependence of protein aggregation
  5. Evidence supporting the importance of α-synuclein steady state levels in PD
  6. Regulation of α-synuclein levels
  7. Concluding remarks
  8. Acknowledgements
  9. References