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

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

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-synuca-synuc-lein 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 causaneurodegenera-tive 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.

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 pathologclin-ical 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-synucc-synuc-lein, 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.

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 elecelec-tric 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 Promopromo-ters 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 Expresexpres-sion 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.

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

57 52 9

1 residues αα-140

E43K

6 5 4

1

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.

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.

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 Perhaps oxidative modification of the protein is more likely to be the cause of the aggregation and metal ions, such as Cu or Fe, can catalyse Fenton chemistry that would generate the oxidative species necessary Finally, is a-synuclein really a copper binding protein under physiological conditions? The low affinities cur-rently reported do not really support this, and there is

no real evidence available to support a functional role

of a-synuclein as a metalloprotein The true situation is that there is insufficient evidence available to conclude one way or the other whether metal binding to a-synuc-lein is an artefact or not However, the new evidence suggesting that a-synuclein is a metal binding protein

is intriguing and is likely to result in a robust, new research direction in the synuclein field

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