Báo cáo khoa học: Interactions between metals and a-synuclein ) function or artefact? pptx

MINIREVIEW Interactions between metals and a-synuclein ) function or artefact? David R. Brown Department of Biology and Biochemistry, University of Bath, UK Introduction Advances in research in recent years have linked many neurodegenerative diseases to specific proteins that undergo either abnormal conformational changes or whose metabolism is somehow modified. Links between Alzheimer’s disease (AD) and amyloid-b (Ab) Creutzfeldt–Jakob disease and prions are well documented [1]. In recent years, another protein has been discovered that is related to a variety of neuro- degenerative disorders. This protein, originally termed the nonamyloid component precursor, was identified in the plaques of AD and was later termed a-synuc- lein [2]. Altered forms of a-synuclein are also found in the deposits termed Lewy bodies (LBs) [3] (Fig. 1). a-synuclein desposits are associated with diseases such as Parkinson’s disease (PD), multiple system atrophy and sporadic and inherited LB diseases. PD is the most common neurodegenerative disorder after AD. LBs can also be identified in some cases of AD. Therefore changes in this protein are associated with the most common neurodegenerative diseases, inclu- ding AD. Due to its recent discovery, research into the causal relation between a-synuclein and these dis- eases remains in its early beginnings. Much of the research related to this protein has been to identify mutations associated with disease [4,5], create an animal model [6] or to understand the mechanism by which the protein aggregates [7]. The function of the protein remains unknown. However, results from knockout mice suggest that it plays an important role in dopaminergic neurones, possibly regulating the release of dopamine from presynaptic termini. Over- expression of a-synuclein results in death of dopamin- ergic neurones of the substantia nigra, further emphasizing the importance of normal regulation of this protein to this cell type [8]. PD is a severe, progressive motor disorder caused by changes in the central nervous system (CNS). True PD is tightly linked to degeneration of neurones in an area of the ventral midbrain or basal ganglia known as the substantia nigra pars compacta. The neurones affected Keywords amyloid; copper; Lewy body; Parkinson’s disease Correspondence D. R. Brown, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK Fax: +44 1225 386779 Tel: +44 1225 383133 E-mail: bssdrb@bath.ac.uk (Received 9 March 2007, revised 1 May 2007, accepted 7 May 2007) doi:10.1111/j.1742-4658.2007.05917.x a-synuclein is one of a family of proteins whose function remains unknown. This protein has become linked to a number of neurodegenera- tive disease although its potential causative role in these diseases remains mysterious. In diseases such as Parkinson’s disease and Lewy body demen- tias, a-synuclein becomes deposited in aggregates termed Lewy bodies. Also, some inherited forms of Parkinson’s diseases are linked to mutations in the gene for a-synuclein. Studies have mostly focussed on what causes the aggregation of the protein but, like many amyloidogenic proteins asso- ciated with a neurodegenerative disorder, this protein has now been sugges- ted to bind copper. This finding is currently controversial. This review examines the evidence that a-synuclein is a copper binding protein and dis- cusses whether this has any significance in determining the function of the protein or whether copper binding is at all necessary for aggregation. Abbreviations Ab, amyloid-b; AD, Alzheimer’s disease; CNS, central nervous system; DLB, dementia with LB; LB, Lewy body; MPTP, 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyrodine; NAC, nonamyloid-b component; PD, Parkinson’s disease. 3766 FEBS Journal 274 (2007) 3766–3774 ª 2007 The Author Journal compilation ª 2007 FEBS are specifically ones that generate the compound dop- amine as a neurotransmitter and are termed dopamin- ergic neurones. The disease was first described in 1817 by James Parkinson and was also termed shaky palsy because of the shaking movement made by the patients. The disease effects approximately 1 in every 500 people. Other diseases with similar symptoms are often des- cribed as ‘parkinsonian’ because of the symptoms exhibited. One such disease is manganism. However, these diseases must be separated from true PD which is specifically a disease result from degeneration of dopaminergic neurones in the substantia nigra. Approximately 50–70% of all the dopaminergic neu- rones are lost from this region before symptoms of the disease appear [9]. Like most neurodegenerative disor- ders, the true cause of the diseases remains uncertain. There is strong evidence that familial or inherited forms are linked to particular point mutations in cer- tain genes such as a-synuclein or parkin. A common treatment of the disorder is to supply l-3,4-dihydroxy- phenylalanine, a precursor molecule for the lost neuro- transmitter. The clinical symptoms of PD include resting trem- ors, muscle rigidity and bradykinesia. As well as exten- sive dopaminergic neuronal loss the presence of LBs, containing a-synuclein fibrils, in the substantia nigra and other brain regions are characteristic of the disease [10,11]. Inclusions containing a-synuclein are also found in dementia with LBs (DLB), multiple system atrophy and the ‘Lewy body variant’ of AD [12]. It is likely that a-synuclein plays a critical role in the patho- genesis of these diseases because rare missense muta- tions in the SNCA gene (resulting in amino acid substitutions A30P, E46K, A53T) or duplication or triplication of the a-synuclein locus have been linked to familial forms of either PD [13–17] or DLB [15]. Furthermore, transgenic animals overexpressing wild-type or mutant human a-synuclein develop clin- ical and pathological features very similar to those observed in PD [18,19], suggesting that the accumula- tion of aggregated forms of a-synuclein in the brain could be the underlying cause of neurodegeneration in PD and related disorders. Most patients with PD have a sporadic form that has not been possible to link to mutations in any known gene. Approximately 15% of patients claim to have a family member who also had the disease [20]. Huge numbers of genetic linkage studies have been undertaken to attempt to find the gene associated with PD in the inherited forms. Paradoxically, there has been no single gene identified as the PD gene. Muta- tions in a large number of proteins have been found. The genetic loci associated with PD have been given the designation PARK. The first of these to be identi- fied was PARK-1 which is associated with the protein a-synuclein and is found on human chromosome 4q21 [13]. In this case, the disease either arises through missense point mutations (A53T, A30P or E46K) or through triplication of the gene. The latter demon- strates that simple increased expression of a-syncuclein could be sufficient to cause disease. These mutations are associate with early onset of the disease and has the pathology includes LBs and is autosomal domin- ant [21]. a-synuclein a-synuclein is a small (14 kDa), highly conserved, presynaptic protein of unknown function, expressed highly in specific brain regions [22–25]. It belongs to a family of proteins including b-synuclein and c-synuc- lein [3]. In contrast to a-synuclein, these two proteins do not appear to aggregate or form fibrils [26]. How- ever, b-synuclein is known to inhibit a-synuclein aggre- gation and the relative levels of the two proteins may be a significant fucator is the occurance of a-synuclein related pathology [27]. a-synuclein has a series of imperfect repeats (KTKEGV) at the N-terminus, as well as an acid C-terminal domain (amino acid residues 96–140) and appear from recombinant studies, to be a natively unfolded protein [23,28–31]. In addition, like most natively unfolded proteins, it has low overall hydro- phobicity and a large net negative charge [32]. a-synuc- leins show 55–62% identity to b- and c-synuclein [3]. a- and b-synuclein have identical N-termini and both these proteins are concentrated in nerve terminals in the proximity of synatpic vesicles [33]. c-synuclein is expressed throughout nerve terminals. a-synuclein Fig. 1. A transverse section of the substantia nigra from a PD patient showing two LBs. These deposits are composed largely of a-synuclein. D. R. Brown Interactions between metals and a-synuclein FEBS Journal 274 (2007) 3766–3774 ª 2007 The Author Journal compilation ª 2007 FEBS 3767 became of interest to the study of neurodegenerative diseases after the discovery of the nature of what was then termed the ‘nonAb component’ (NAC) of plaques in AD [34,35]. The protein then termed NAC-precur- sor or phosphoneuroprotein-14 turned out to be a homologue of synuclein originally identified in the elec- tric organ of the Pacific electric ray (Torpedo californi- ca) [10]. Subsequenty, there has been no conformation of a role of NAC in AD but a-synuclein is now accep- ted as the main component of LBs as found in PD and LB dementias [Fig. 2]. a-synuclein can also be found as small oligomers or smaller aggregates associ- ated with synapses and it is possible these forms con- tribute to the disease process rather than LBs [36]. a-synuclein can bind to lipids membranes through its N-terminal repeat region [37] and can selectively inhibit phospholipase-D2 [31]. This phospholipase is localized to the cell membrane where it is involved in signal-induced cytoskeleton regulation and endocytosis. It is therefore possible that alpa-synculein regulates vesicular transport processes. a-synuclein appears to be phosphorylated [38] and this may have some conse- quences for the protein’s function. There is some evi- dence that the protein interacts with synphilin-1 [39], another protein of unknown function, and this protein has also been identified in LBs [40]. Knockout mice have been generated that do not express a-synuclein. These mice do not show any neuropathological changes, suggesting that loss of function of the protein does not play a direct role in any form of cell death [41]. However, loss of the pro- tein does result in abnormal activity of dopaminergic neurones in substantia nigra, with reduced levels of dopamine detected in the striatum. This implies that the protein could play a role in the regulation of neutransmitter release. A second strain of a-synuclein knockout mice was also developed [42]. Certain toxins will induce parkinsonian changes in mice. One such compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyro- dine (MPTP) induced degeneration and loss of dopam- inergic neurones. This second line of knockout mice proved resistant to the effect of MPTP. MPP+, the metabolic product of MPTP, acts on various elements of the synaptic machine. Again, these results suggest a role for a-synuclein in vesicular function. Recently, a double knockout mouse has been generated lacking both a- and b-synuclein [43]. Again dopamine levels were found to be reduced in the brain but studies of neurones isolated in culture found no differences to wild-type mice. This suggests that synucleins are not essential components of the machinery that causes neu- rotransmitter release, but they may contribute to long- term regulation of presynaptic activities. Given the similarity between the synuclein, possibly a triple knockout mouse is necessary to understand the func- tion of these proteins in the CNS. It is more likely, however, that the role played by a-synuclein in disease results from dysfunction due to its aggregation. There have been more than ten groups that have generated a-synuclein transgenic mice [18,44–52]. The mice either expressed human wild-type a-synuclein or the human protein carrying one of the two main mutants (A53T or A30P). These mice differ in the level of expression of the protein and in the kind of promo- ter used to generate expression. Promoters used include those for platelet-derived growth factor-b, Thy-1, prion protein, tyrosine hydoxylase or an oligo- dendrocyte specific promoter. The results from these many experiments were quite variable. However, many of the mice produced accumulations of a-synuclein and showed changes in the dopaminergic system. In addition, many of the mice showed motor changes reminiscent of the parkinsonian tremor or altered loco- motion or coordination. However, none of the mice showed neuronal loss, no matter how high the expres- sion level or the presence of mutations. Expression within glial cells resulted in inclusions with a greater similarity to LBs. The failure of transgenic mice to result in a reliable model of PD is possibly due to the expression of a human protein in a mouse. Co-expres- sion of mouse and human a-synuclein could alter the ability of the human protein to form a toxic molecule, Fig. 2. Cell inclusions: overexpression of a-synuclein in cells results in aggregation of a-synuclein within the cells. This SH-SY5Y over- expressing a-synuclein was immunostained for a-synuclein. In approximately 10% of cases, these aggregates form on a large single aggregate resembling an LB. Image supplied by Josephine Wright. Interactions between metals and a-synuclein D. R. Brown 3768 FEBS Journal 274 (2007) 3766–3774 ª 2007 The Author Journal compilation ª 2007 FEBS as it is known that mixing human and mouse a-synuc- lein inhibits the ability of the protein to aggregate [53]. Therefore, the animal models based on neurotoxins such as MPTP and rotenone are more reliable and reproduce the disease more effectively than transgenic mice. Unfortunately, such models do not provide insight into how changes in a-synuclein cause disease as the disease is generated by another source. Recently, a viral vector system was used to directly transfer the human a-synuclein into the substantia nigra of a rat [54]. The recombinant adeno-associated viral vector resulted in high expression of the human protein in the substantia nigra and, after 13 weeks, the research- ers observed a 50% reduction in the number of dopam- inergic neurones. Unlike other models, the progression of cell loss was slower, more like PD. In addition, there were other changes that were more similar to the human disease, including phosphorylation of a-synuclein at serine 129 and activation of caspase 9. This system possibly represents a better model of PD. Metal binding The study of a-synuclein function has been hampered by a lack of phenotype in knockout mice and the inconclusive nature of studies from cell biology and bio- chemistry. Although studies do suggest that a-synuclein expression can influence a variety of cellular activities such as vesicle trafficking, no study has clearly shown that the protein is essential for anything. Although it might be easy to conclude that the protein has no func- tion, it is rather nonsensical to do so because the protein is evolutionarily conserved and has homologues such as b-synuclein. One of the simplest recourses for studies of function is to associate the protein with particular cofactors that are commonly used for a variety of biological activities. With the suggestion that a-synuclein binds copper, we have the potential to link the protein into copper metabolism or activities associated with copper binding proteins. Initial evidence for the potential of a-synuclein to bind metals came from the ability of certain metals catalyse aggregation of the protein (see below). It was suggested the protein could bind up to ten atoms of copper with a low affinity value of 59 lm [55]. Analy- sis of the potential binding site indicated that aggrega- tion of the protein was only initiated if the C-terminus was intact [56]. This suggested that the C-terminus was the principal binding site for Cu (Fig. 3). Despite early studies suggesting the Cu causes aggregation of the protein via the C-terminus, a more recent study sug- gests that Cu binds to the histidine at residue 50 in the N-terminus with higher affinity [56]. Binding at the high affinity site was shown to be sufficient to drive oligomerization of the protein. The high affinity site appeared to be a type-II copper binding site with square planar co-ordination. However, the affinity for this site is suggested to bind Cu at 0.1 lm. That for the second site was shown to bind Cu at 50 lm, which is in line with previous findings [55,57]. However, these affinities seem rather low for an intracellular protein where Cu would be bound by proteins with a much higher affinity. Under such conditions, it is likely that these other proteins would out-compete a-synuclein for Cu. The C-terminus of the protein has also been sug- gested to bind polyamines [58]. These polyamines can also initiate aggregation of the protein. Different papers suggest Cu initiates aggregation at the C- and N-terminal copper binding sites. These conflicting reports and the weak affinity constants mean that whe- ther Cu binds or not and where is uncertain. It is poss- ible that the weak affinity measures simply reflect deficiencies in the experimental design but, clearly, fur- ther investigation is necessary. Despite these deficiencies, further research on Cu binding has continued. The results from some more recent papers are as intriguing as they are confusing. Although a previous study [56] suggested that only the histidine at position 50 was necessary for Cu binding, a more recent study suggested that Cu binding requires nine N-terminal amino acid residues in addition to amphipathic region Seven II-mer repeats NAC 7 89 140 57 52 9 1 residues α α -140 A30P A53T E43K High affinity Cu Site Low affinit y Cu Site 65 4 231 acidic tail (capable of forming 5 α-helixes) Fig. 3. Linear representation of the a-synuc- lein protein. The location of the repeats in the N-terminus, the NAC domain thought to be involved in aggregation and the proposed Cu binding domains are shown. Also shown are the locations of the three mutations associated with inherited froms of PD. D. R. Brown Interactions between metals and a-synuclein FEBS Journal 274 (2007) 3766–3774 ª 2007 The Author Journal compilation ª 2007 FEBS 3769 residues 48–52 [59]. Furthermore, it was suggested that the C-terminal site (residues 191–124) binds other metals (such as Mn or Fe) with a very low (mm) affin- ity. Such low affinities should really be considered as nonspecific associations and do not provide convincing evidence for a-synclein being a metal binding protein. In a further NMR study, a-synuclein was suggested to have as many as 16 different amino acid residues that could participate in Cu binding [60]. Deletion of H50 from the protein did little to abolish Cu binding and clearly showed that this histidine may participate in Cu binding but is not critical. Although proposing additional C- and N-terminal binding sites, the study did not provide conclusive evidence for a high affinity Cu site but suggested that a loose association between many amino acids resides and Cu was possible. As a variety of amino acids can bind Cu in solution, this result is neither surprising, nor convincing. Two further studies with peptide fragments of the N-terminus have been published. These studies are lar- gely based on potentiometric techniques augmented with circular dicroism and electron paramagnetic reso- nance spectroscopies. They showed that aspartate in the first 17 amino acid residues plays an important role in the co-ordination of Cu [61]. This interaction can also result in the oxidation of methionine residues [62]. However, using similar techniques, the same group showed that Cu is co-ordinated with a peptide with residues 39–56 and that both the histidine and lysine res- idues are involved in the co-ordination [63]. Although these studies provide interesting insights into the co-ordination of Cu by these fragments, they do not provide any more convincing evidence that these Cu–peptide interactions are specific and would occur in vivo. Additionally, Cu-peptide studies are notori- ously misleading because Cu co-ordination changes with large fragments and it is possible that full length a-synuclein would bind Cu in a completely different way to these artificial peptides. Although the summation of these various studies points clearly towards a future for a-synuclein as a metalloprotein, the evidence necessary to make the story convincing remains undiscovered. Further studies carried out under physiological conditions could potentially provide the missing element needed to demonstrate that Cu binding to a-synuclein is not an artefact. a-synuclein aggregation Cells expressing high levels of a-synuclein have been shown to generate aggregates of protein [64] (Fig. 2). It remains unclear as to why this occurs and what the mechanism is behind the process. It is also unclear whether this process is really causal to neurodegenera- tive disease. The formation of LBs or other aggregates of this protein might be a result of the disease rather than the cause. However, as inherited mutations in the protein are associated with PD, it is likely that the pro- tein plays some role in the progression of the disease. Both peptides and recombinant protein have been used to study the aggregation properties of a-synuc- lein. Filaments will form from the N-terminal domain of the protein with similar properties the protein aggregates extracted from the brains of Parkinson patients [4,65–68]. The kinetics of fibrillation are con- sistent with a nucleation mechanism [69,70]. The key step in the transformation of the protein to a form able to aggregate involves a partially folded interme- diate [64]. The final transition of the protein results in a gain of b-sheet content. The fibrils that are formed are amyloid-like, around 5–10 nm in length with a diameter of 4–8 nm (Fig. 4). These can cluster together to form bundles of 50 nm and up to 1 mm in length [2,66,71]. Peptides from amino acid resi- dues 1–18 behaved similarly to the full length protein, suggesting that this domain is key to the aggregation. In comparison, a peptide based on amino acid resi- dues 19–35 remained soluble and unstructured [72]. Further analysis suggested that the residues 8–16 are key to the formation of b-sheet. These findings are in contrast to another study suggesting that the main site regulating protein aggregation is around amino acid residues 64–100 [73]. Small peptides from this region (residues 69–72) have been shown to block aggregation of the full-length a-synuclein [73]. It is possible that both these regions add to the fibril for- mation of the protein. Fig. 4. a-synuclein can aggregate to form fibrils. Electron micro- scopic analysis of purified a-synuclein fibrils shadow stained with phosphotungstic acid. a-synuclein forms long filamentous struc- tures when it aggregates under specific conditions and such fil- ments can also be extracted from LBs. Interactions between metals and a-synuclein D. R. Brown 3770 FEBS Journal 274 (2007) 3766–3774 ª 2007 The Author Journal compilation ª 2007 FEBS Of greater interest is the assessment of factors that could contribute to the aggregation of the protein. Phosphorylation of serine 129 increases fibril formation [74]. Sequence modification is the most obvious cause of increased aggregation and mutations associated with pathology in particular [75]. In addition, oxidation and nitration can also increase the rate of conversion [76]. Binding of polyanions to the C-terminal domain can also catalyse protein aggregation [58]. Current research has suggested that one of the main factors affecting self-oligomerization of the protein is the presence of metals, such as Cu. Cu has been sug- gested to be the most effective metal in terms of inducing oligomerization [55]. Cu chelators were shown to abolish aggregation. This oligomerization seemed to be mediated by interaction of Cu with the C-terminus of the protein. This was shown by limited proteolysis of the a-synuclein that cleaved off part of the C-terminus, either at residue 97 or 114. The shorter fragment showed no response to Cu in terms of oligomerization, whereas the 1–114 fragment did produce a limited amount of oligomerization [57]. Further studies showed that metals not only induce aggregation, but also induce conforma- tional change. Aluminium was found to be the most effective metal at induction of polymerization with Cu and Fe being similarly effective [30]. Analysis suggested that the mechanism of polymerization could either lead to amorphous aggregates or structured fibrils. Structural analyses also showed that the metals induced a switch from unstructured to a b-sheet structure. The concentration of metals necessary to produce a-synuclein aggregation is quite controversial. In general, concentrations of metals shown to cause aggregation or fibril formation of a-synuclein are well above physiological values. In other words, the con- centrations would be toxic on their own. Although one study [56] has suggested that as little as 40 lm can initiate a-synuclein aggregation, this has not been dem- onstrated in others [56,65]. Also, this concentration is higher than the concentration of the ‘high’ affinity binding site reported by the same authors [56]. There- fore, it is unclear whether metal induced aggregation results from specific binding of a metal or a nonspecific association. If the latter is the case, then it is quite possible that the metals themselves are unnecessary for the process and all that is really required is oxidation, as suggested by other studies [77]. Conclusions There is no doubt that a-synuclein is associated with neurodegenerative disease. The fact that mutants of a-synuclein are associated with inherited forms of PD provides clear evidence for this. However, the mechan- ism or role of the protein remains elusive. Is aggrega- tion important for its effect or just high levels of expression? Similarly, aggregates of a-synuclein in the form of LBs are associated with CNS disease, but is aggregation really caused by binding of particular metal ions? The very high concentrations of metals that have been shown to effective in vitro do not really support this. 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