Aggregation and properties of α-synuclein and related proteins

α-Synuclein has been identified as a component of intracellular fibrillar protein deposits in several neurodegenerative diseases, and two mutant forms have been associated with early onset Parkinson’s disease. A fragment of α-synuclein has also been identified as the non-A β component of Alzheimer’s disease amyloid (NAC). Ageing solutions of α-synuclein and NAC leads to formation ofβ-sheet, detectable by circular dichroism spectroscopy, and aggregation to form amyloid-like fibrils, detectable by electron microscopy. Differences in the rates of aggregation of the fibrils formed by α-synuclein and the two mutant proteins are presented. The toxicity of α-synuclein and related peptides towards neurons is also discussing in relation to the aetiology of neurodegenerative diseases. Experiments on fragments of NAC have enabled the region of NAC responsible for its aggregation and toxicity to be identified.


Introduction
Synucleins are a family of small proteins (127-140 amino acid residues for the human forms) expressed at highest levels in nervous tissue.Three members, α-, β-, and γ-synucleins, are the products of three genes present on three different chromosomes [10].A fourth member, synoretin, is expressed most highly in retina [49].The first indication of an involvement of α-synuclein in the pathogenesis of neurodegenerative diseases came from the isolation, from purified amyloid of Alzheimer's disease brains, of a novel peptide unrelated to Aβ.This peptide, representing about 10% of the non-SDS soluble material, was named non-Aβ-component of Alzheimer's disease amyloid (NAC).Sequencing revealed that NAC comprised at least 35 amino acids, although the N-terminal residues could not be assigned with certainty because of the specificity of the enzyme used in sequencing [53].These 35 amino acids correspond to residues 61-95 of a 140 amino-acid precursor (NACP), later shown to be identical to α-synuclein.Interest in α-synuclein was enormously enhanced when two different mutations in the α-synuclein gene were found in inherited forms of Parkinson's disease.One mutation, α-synuclein(A53T), found in certain Italian and Greek families, results in an Ala 53 to Thr substitution in a region predicted to adopt an α-helical structure surrounded by β-sheets [42].The other mutation, α-synuclein(A30P), an Ala 30 to Pro change, was detected in a family of German origin [32].It has been suggested that these amino acid substitutions may disrupt local α-helical structure, extending the β-pleated sheet and so rendering mutant α-synuclein more prone to self-aggregation [32,42].This might lead to the formation of abnormal deposits in the brain, in an analogous manner to the accumulation of protein aggregates such as amyloid β-peptide (Aβ) or tau in Alzheimer's disease, huntingtin protein in Huntington's disease, and prion protein in the transmissible spongiform encephalopathies.
Lewy bodies and Lewy neurites constitute the main pathological features in the brains of patients with Parkinson's disease and dementia with Lewy bodies.Lewy bodies and Lewy neurites contain α-synuclein in a fibrillar form [3,45,46].Thus, these α-synuclein fibrils could be analogous to the insoluble protein aggregates found in other forms of neurodegenerative disease.Additional immunohistochemical and immunoelectron microscopy studies have shown that α-synuclein is also associated with pathological lesions in other neurodegenerative diseases involving non-neuronal cells, such as the glial cytoplasmic inclusions found in multiple system atrophy [34,47,55].Several neurodegenerative diseases involving α-synuclein, collectively known as synucleinopathies, are discussed in a recent review [51].
Protein conformation-dependent neurotoxicity is an emerging theme in neurodegenerative disorders such as Alzheimer's disease, Huntington's disease and prion disease [33].A similar conformationdependent mechanism may also be relevant to the synucleinopathies.Thus there have been several studies into the aggregation of α-synuclein, its mutants and NAC, and attempts to determine what structural features govern this behaviour.We describe below studies on the aggregation of NAC and α-synuclein, wild-type and mutant forms, as well as conformational changes associated with fibril formation.Toxicity of these proteins to cultured cells is also discussed.

Aggregation of NAC
Since NAC was originally isolated from an amyloid preparation it was not surprising that subsequently NAC was found to aggregate in vitro [27,31].These aggregates have been shown, by thioflavin-S staining, Congo Red staining and Fourier-transform infrared spectroscopy, to contain β-sheet structure, indicative of the presence of amyloid-like fibrils.Electron microscopy revealed the presence of clumps of short irregular fibrils of variable length, mainly of diameter 4-11 nm, similar in size to those found in neuritic plaques [17,18,27,31].A representative example of fibrils formed by NAC is shown in Fig. 1.
Changes in secondary structure when solutions of proteins are aged (i.e., incubated, usually at 37 • C, for varying times) can be quantified by circular dichroism spectroscopy.A transition from totally random structure to predominantly β-sheet upon ageing of NAC in solution can be seen in Fig. 2 [17,18].The formation of amyloid can be revealed by binding to thioflavin-T, with consequent increase in fluorescence.The rate of amyloid formation by NAC using this assay is shown in Fig. 3.
To pinpoint the exact region responsible for NAC aggregation we repeated similar experiments on smaller fragments of NAC.NAC(8-18) (residues 68-78 of α-synuclein) was the smallest fragment that  aggregated, as indicated by concentration of peptide remaining in solution after 3 days, and formed fibrils, as determined by electron microscopy [8].The importance of the NAC region in controlling aggregation of α-synuclein has also been emphasised by other workers.Consideration of the primary structure suggested residues 72-84 in α-synuclein were critical for its more rapid aggregation compared to β-and γ-synucleins [6].A peptide comprising residues 71-82 of α-synuclein has also been shown to aggregate [26].We believe that the fact that there is only partial overlap between the sequence defined by our work and that suggested by these other groups is a consequence of the reductionist approach we adopted.We investigated smaller and smaller fragments until we were able to define the minimum required sequence.
It should be noted that the presence of NAC in Aβ-containing plaques from Alzheimer's disease patients has been disputed by other groups who were unable to show immunostaining of plaques by antibodies to the NAC region [4,14].It is possible that some of the confusion may arise from the diagnosis as Alzheimer's disease of cases of Lewy body variant of Alzheimer's disease.For example, α-synuclein accumulates in dystrophic neurites that decorate the core of Aβ plaques from patients with Lewy body variant, but not in comparable material from typical Alzheimer's disease patients [57].

Aggregation of α-synuclein
α-Synuclein comprises 140 amino acids in two domains, linked via the NAC sequence.The C-terminal domain contains many acidic amino acid residues.It can undergo post-translational modification, including phosphorylation of Ser 87 and Ser 129 [37] and Tyr 125 [21].The N-terminal domain, which is highly conserved between species, comprises 7 repeats of a degenerate 11-amino-acid motif.This feature is reminiscent of many apolipoproteins that form amphipathic α-helices [23], and indeed α-synuclein can bind to small acidic phospholipid vesicles, resulting in a marked increase in α-helicity [15].Experimental confirmation that it is indeed the N-terminal domain of α-synuclein that binds lipid and increases its α-helicity has been obtained by NMR spectroscopy [20].However, in its native state α-synuclein is unfolded and may function as a chaperone protein.It is also much more soluble than is NAC [56].Indeed it was only after the link to Parkinson's disease was discovered that α-synuclein was shown to be capable of aggregating to form amyloid-like fibrils similar to those that have been isolated from Lewy bodies [28].
Although it is clear that two mutations in the α-synuclein gene can give rise to an inherited form of Parkinson's disease, the mechanism by which the disease occurs is not known.One possibility is that α-synuclein containing these mutations aggregates more rapidly, and one or both α-synuclein mutations have been reported to accelerate the aggregation process [11,16,24,35].Aggregates formed from wild-type α-synuclein or from either mutant were thioflavine-S positive, indicative of the presence of aggregates with the β-pleated sheet conformation characteristic of amyloid fibrils [16,28].Anti-parallel β-sheet structure in wild-type and mutant aggregates has been confirmed by Fourier transform infrared spectroscopy [13,35].
We found that self-oligomerisation could be detected by silver staining after SDS-PAGE of freshly dissolved or aged samples of wild-type and mutant α-synucleins [19].Larger amounts of dimeric species and oligomers were present in aged than in fresh solutions.Furthermore, increased amounts of high molecular weight species that did not enter the separating gel were found in aged samples, suggesting the formation of larger aggregates.α-Synuclein aggregation is temperature, concentration, and timedependent [24,28,35].More recently, the kinetic mechanism of α-synuclein aggregation was studied and found to be rate limited by a nucleation step.Addition of preformed fibrils of α-synuclein caused rapid aggregation of a supersaturated solution, bypassing a lag phase that occurred in the absence of seeding.Aggregate growth followed first-order kinetics with respect to α-synuclein concentration, and α-synuclein(A53T) could seed the aggregation of wild-type α-synuclein.In addition, the wild-type and mutant forms of α-synuclein had similar critical concentrations, as measured by peptide remaining in solution after complete aggregation and attainment of equilibrium between peptide in solution and peptide in fibrillar form.These results led the authors to suggest that the more rapid rate of aggregation of A53T α-synuclein compared to wild-type could not be explained by decreased solubility but was due to increased nucleation rate [58].A partially folded form of α-synuclein, stabilised by low pH and high temperatures, has been detected and may be an intermediate in the conversion between monomer and oligomeric forms [54].A variety of non-fibrillar oligomers, including protofibril-like chains of spheres and rings that may represent circularised protofibrils, has been detected by atomic force microscopy.The disappearance of the monomeric form of the A30P mutant in the absence of fibril formation may reflect formation of such non-fibrillar forms [12].Some differences in the morphology of fibrils formed by wild-type and mutant α-synucleins have been reported [16,24].We used electron microscopy to examine uranyl acetate-stained fibrils preparations, made from aggregated samples of α-synucleins [16].Fibrils of variable length and 5-25 nm in diameter from aged samples of α-synuclein proteins, similar in size to those found in Lewy bodies and Lewy neurites were detected.In general, the mutant synucleins produced fibrils of greater diameter than did wild-type.
The structures of wild-type and mutant α-synuclein proteins in solution have been studied using circular dichroism spectroscopy.α-Synuclein in freshly prepared solutions was found to exist in random conformation, a condition that has been described as "natively unfolded" [56].However, ageing a solution of α-synuclein(A53T) led to a change in secondary structure from predominantly random to a mixture of β-sheet and random conformations [11,16].In contrast, when a similar experiment was carried out on wild-type and mutant α-synuclein(A30P) under similar conditions, the spectrum of both proteins showed less change upon ageing [12,16].The fact that we did not directly observe a transition to β-sheet during ageing and aggregation of wild-type α-synuclein and the mutant α-synuclein(A30P) may be due to the particular kinetics of aggregation for these proteins [16].The use of the membranemimicking solvent aqueous acetonitrile, which is known to stabilise β-sheet [18,22,36,59] emphasises the structural differences between wild-type and mutant α-synucleins [16].Freshly prepared solutions of α-synuclein mutants in acetonitrile/phosphate buffer saline mixtures (1 : 1) exhibited β-sheet conformation.The β-sheet content of mutant α-synuclein(A53T) was even higher than that of the mutant α-synuclein(A30P) as suggested by the intensity of the circular dichroism spectra.In contrast, wild-type α-synuclein under the same conditions gave a spectrum with a mixture of random and β-sheet conformations.These results would suggest that structural transition from random coil to predominantly β-sheet must occur as a prelude to aggregation for wild-type and mutant α-synucleins.Such a transition in secondary structure may well be a general prelude to the formation of toxic fibrils by amyloidogenic proteins, as has been suggested previously [33].

α-Synuclein toxicity
The similarity between NAC and other amyloidogenic proteins that contain neurotoxic regions, with respect to their sequence and their propensity to form fibrils consisting of β-sheet, led us to believe that NAC might also be neurotoxic [18].Although the existence of extracellular NAC remains controversial, there have been recent reports that α-synuclein is present in CSF [9] and in extracellular Lewy bodies [50].If α-synuclein accumulates in neurons, which eventually die, aggregates could leak out of the dead neuron and spread the disease to neighbouring cells [34].Moreover, α-synuclein has been shown to coexist with tau in neurons in Alzheimer's disease [2] diffuse Lewy body disease [30] and multiple system atrophy [40].The toxicity of α-synuclein and related peptides may thus have pathological relevance in several neurodegenerative diseases.On this basis we investigated the in vitro toxicity of wild-type and mutant α-synucleins and NAC-related peptides [17].
We also investigated the mechanism involved in the toxicity induced by α-synucleins and NAC peptides.Morphological examination of cell nuclei, stained with the DNA-binding fluorochrome Hoechst 33258, showed that several cells exposed to α-synucleins or α-synuclein fragments NAC and NAC(1-18) presented a typical apoptotic morphology, including condensation of chromatin and nuclear fragmentation [17].
There have been several reports that implicate oxidative damage to the aetiology of neurodegenerative diseases.Incubation of α-synuclein in an oxidative system derived from cytochrome c and hydrogen peroxide increased the rate of aggregation of the former protein.Moreover, cytochrome c and α-synuclein were shown, by double labelling, to be co-localised in Lewy bodies from brains of Parkinson's disease patients [29].Oxidative damage, in the form of nitrated α-synuclein in Lewy bodies, has been directly demonstrated using an antibody that recognised the nitrated protein [25].Overexpression of α-synuclein is toxic to neurons [38,43].Iron and free radical generators stimulate the production of intracellular aggregates of α-synuclein [39].NAC and α-synuclein, but not β-or γ-synucleins, liberate hydroxyl radicals when incubated with iron(II) [52].
Inclusions containing ubiquitinated proteins have been detected in several neurological disorders and ubiquitin has been used as a marker for Lewy bodies.Ubiquitin tags proteins earmarked for degradation by the proteasome.It has been suggested that failure to degrade ubiquitinated protein aggregates may be part of the pathology of neurodegenerative diseases [1] and indeed it has been shown that inhibition of proteasome activity results in decreased α-synuclein turnover [5].In a recent significant development, a link has been discovered between two proteins, parkin and α-synuclein, that have mutant forms known to be implicated in Parkinson's disease.Normal parkin is an E3 ubiquitin ligase, and a mutation in this protein is associated with autosomal recessive Parkinson's disease.Normal parkin, but not the mutant type, forms a complex involving a glycosylated form of α-synuclein.Defective parkin thus resulted in accumulation of non-ubiquinated synuclein [44].

Fig. 3 .
Fig. 3. NAC(1-35) aggregation as monitored by thioflavin T fluorometric assay.The increase in thioflavin T fluorescence is a measure of the concentration of aggregates with β-sheet conformation, present after incubation of a solution of peptide (1 mg/ml) at 37 • C. For each data point, error bars indicate 95% confidence limits.

Fig. 5 .
Fig. 5. Aggregation of peptides NAC(1-18) (filled diamond) and NAC(19-35) (filled square).Peptides (0.4 mM) in PBS containing 0.025% sodium azide, were incubated at 37 • C and aliquots assayed at intervals over 72 h.Each data point represents the mean of at least four independent experiments.Standard deviations are shown for each point as bars above or below the mean; in some cases the spread is smaller than the symbol used and bars are not therefore visible.Statistical comparisons between the behaviour of the two peptides were performed using Student's T -test.Significant differences are indicated by ** (p < 0.001).This figure is reproduced from El-Agnaf et al. [18] and reprinted with permission from Blackwell Science, Oxford, UK.