ASynuclein And Parkinsons Disease

The Parkinson's-Reversing Breakthrough

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Parkinson's disease (PD) is the most common age-related neurodegenerative movement disorder [215]. The primary symptoms of PD are caused by the loss of dopaminergic neurons in the substantia nigra region of the brain stem [216]. A diagnostic hallmark of PD is the presence in the cerebral cortex of intracellular inclusions (Lewy bodies and neurites) [217,218], but the role of these proteina-cious materials in the pathogenic process has not been established. a-Synuclein (a-syn), an abundant 140-residue neuronal protein of unknown function [219], is the primary component of the fibrillar inclusions. Autosomal dominant early-onset PD has been linked to two point mutations (A53T and A30P) in the gene encoding a-syn [220,221]. Introduction of human wild-type, A30P, or A53T a-syn into transgenic animal models produces age-dependent motor dysfunction, neuronal deposits of fibrillar protein, and loss of dopaminergic neurons, consistent with suggestions that PD may arise from these protein aggregates [222,223].

In vitro fibrillogenesis experiments also have shown that the two mutants aggregate faster than the wild-type protein [224,225], consistent with the proposal that the disease is caused by protein aggregation. Fibrillar a-syn, however, may be a symptom rather than a cause of the disease. Recent reports suggest that the formation of soluble oligomeric intermediates is accelerated in the A30P mutant [226]. Although there are contradictory results, the A30P mutant has been reported to bind more weakly to lipid vesicles than the wild-type protein [227-230]. These observations point to a role for prefibrillar species in PD [226,231] and, further, that a-syn interaction with synaptic-vesicle membranes may be involved in the pathogenic mechanism [232,233]. Indeed, it has been proposed that fibrillar aggregates are a byproduct of neuronal death and that the formation of Lewy bodies could be a protective mechanism to sequester neurotoxic species [234-237].

a-Synuclein is localized in the cytosol and presynaptic terminals of neurons, with some fractions associated with synaptic-vesicle membranes [238-241]. Although its function has not been determined definitively, suggestions include a role in neuronal plasticity and synaptogenesis [238,242] as well as a protein folding chaperone [243]. The amino-acid sequence includes seven imperfect repeats in the N-terminal portion that are similar to an 11 residue repeating motif in exchangeable apolipoproteins [238]. This similarity suggests that the protein may be capable of reversibly binding to the surfaces of lipid membranes [228,229,232,244]. A distinguishing characteristic of the amino-acid sequence is the highly acidic C-terminal region. In vitro, a-syn has been characterized as a monomeric, intrinsically unstructured (natively unfolded) protein [245]. Small-angle X-ray scattering studies indicate that the protein has a radius of gyration (Rg ~ 40 A) that is larger than expected for a folded globular (15 A) polypeptide [246]. However, in the presence of acidic phos-pholipid vesicles, the protein undergoes a conformational change, forming some a-helical structure observable by CD [244] and NMR [247-249], as well as by EPR [250,251]. Interestingly, in contrast to the monomeric protein, larger oligomeric intermediates form p-sheet structures that cause membrane leakage in vitro [233,252], providing further support for the hypothesis that prefibril-lar structures may be cytotoxic. Large environmentally induced conformational changes of this type are particularly interesting. Toxic conformers of otherwise benign proteins have been invoked as pathogens in a number of neurodegenera-tive diseases [253,254].

We have employed FET kinetics to probe the structure and dynamics of pseudo-wild-type and disease-related (A30P) a-synucleins [255]. A fluorescent amino acid (tryptophan) and a chemically modified tyrosine (3-nitrotyrosine, Y(NO2)) were chosen as DA; this pair has a ro of 26A [256]. Tryptophan fluorescence decay and energy transfer kinetics were used to characterize the conformational heterogeneity of the protein under a variety of solution conditions.

Trp residues were incorporated at the sites of three different aromatic amino acids (F4, Y39, F94) in a-syn; in buffer all three variants exhibited emission properties characteristic of water-exposed indole side chains. In the presence of SDS micelles, Trp4 and Trp39 emission maxima exhibited a pronounced blueshift indicative of a more hydrophobic micellar environment that is consistent with previous NMR studies [247-249]. Time-resolved fluorescence polarization measurements also revealed an increase in the microenvironment viscosity and rigidity of the polypeptide structure in the presence of SDS micelles.

In order to probe the equilibrium state of aggregation in solution, Trp fluorescence decay kinetics were examined at different a-syn concentrations. For Trp39 and Trp94, the observed kinetics were independent of protein concentration, whereas Trp4 fluorescence decay kinetics exhibited a modest dependence on [a-syn], suggesting some type of interprotein interaction at protein concentrations above 20 ^M. To explore possible N-terminal interpeptide interactions, FET kinetics of mixtures of D-only and A-only proteins (F4W and Y39(NO2); Y39W and Y39(NO2)) were measured; however, there was no evidence for interprotein contacts in the 3-15 ^M concentration range. It is clear that the state of protein aggregation within the low micromolar concentration range is constant and that, most likely, only monomeric species are present. Moreover, Trp fluorescence decay provides no evidence to suggest that the A30P mutation induces a change in the aggregation state of the protein at concentrations below 30 ^M.

Since a-syn has been characterized as a natively unfolded protein, FET kinetics were fit to DA distance distributions for freely jointed polymer chains [100]. Although this model does not capture all the structural features, the results provide an approximate description of the conformational heterogeneity in terms of a well-defined Gaussian chain model (Figure 21). As expected, the mean DA distances scale with the number of residues separating D and A. Also, from this model an effective length parameter (l') can be extracted which is related to the stiffness of the chain [100]. For a freely jointed polypeptide, the length of the chain segment is generally taken to be the length of a residue of 3.8 A. Stiffer polypeptide chains will have larger values of I'. At physiological pH, five of the DA pairs have chain segments in the 13-16 A range, corresponding in each case to 3-4 amino-acid residues. Under acidic conditions, where a-syn aggregation is accelerated [257], the chain segments in the N-terminal region of the protein lengthen slightly, whereas the C-terminal DA pair exhibits a dramatic decrease in chain segment length (~2 amino-acid residues) at pH 4.4. Apparently, the neutralization of the negative charges in the C-terminus produces a relatively flexible polymer that behaves like a Gaussian coil. Upon addition of SDS, CD spectra indicate that our mutant a-synucleins adopt ~65% helical secondary structure (CD, 222 nm). However, the fits do not reveal a consistent trend in segment lengths in SDS. The greatest effect appears as a substantial increase in segment length (~5 residues) in the C-terminal region of the protein.

W4-Y19

Y19-W39

W4-Y39

A 1

Y74-W94

W94-Y136

.-■■í" 1

W4-Y136

-r

1

Figure 21. Tryptophan to 3-nitrotyrosine distance distributions for freely jointed polymer chain model (dotted line) and DA distributions directly extracted from FET kinetics (solid line) of a-synuclein under physiological pH conditions.

Fits to the FET kinetics data reveal that the Gaussian chain distance distribution is just a rough approximation to the conformational heterogeneity of a-syn. DA distances also were extracted directly from the Trp fluorescence decay kinetics using a linear least squares procedure without recourse to a specific polymer model [48]. This fitting procedure produces the narrowest distance distributions required to fit the data. In all cases, the LLS method gave better fits to the data than the Gaussian chain model, but the general trends in the fitting results were comparable. The distance distributions projected from Trp decay kinetics in pH 7.4 solutions reveal both compact and highly extended conformations. For proteins with the DA pair separated by 15 and 20 residues, regardless of location in the sequence, the protein ensemble includes short (15Á; < 10%), intermediate (~20Á; ~45%), and extended (> 30Á; ~45%) polypeptides (Figure 21). As expected, increasing the number of residues between DA pairs shifts the majority of the population to distances beyond > 40Á.

In the presence of SDS, average DA distances decrease, particularly for the Y19-W39 and W4-Y39 pairs. Based on chemical shifts in Ca NMR spectra, residues 18-31 are expected to have the highest degree of a-helical structure [247]. As found in the Gaussian chain fits, the distance between the W94-Y136 pair increases significantly in SDS, presumably owing to repulsive electrostatic interactions with the negative micelle surface. The most striking feature of the a-syn structures in the presence of SDS micelles is the lack of tertiary contacts; there is no evidence for globular structure despite the development of a-helical secondary structure. Although a-syn has substantial helical content in the presence of micelles, the FET data reveal that the protein remains highly disordered and largely extended.

Acidic conditions do not change the general character of the six DA distance distributions, but there is a slight increase in the amplitudes of the shorter distance populations. Our results are consistent with small angle X-ray scattering studies that indicate the average radius of gyration shrinks from 40 at pH 7 to 30 A at pH 3.0 [246]. The greatest structural change induced by acidic conditions is the contraction in the C-terminal region. The correlation with accelerated aggregation under acidic conditions [246] raises the possibility that the high charge and extended conformation in the C-terminus serve to inhibit aggregation. When the acidic side chains become neutralized, reduced electrostatic repulsion induces shortening of the C-terminus and overall collapse of the polypeptide chain. Notably, C-terminally truncated a-syn (1-110) aggregates faster than the full-length protein [258,259].

FET kinetics of two W/Y(NO2) pairs in the vicinity of one of these PD related mutations (A30P) were examined as functions of solution conditions. Interestingly, the presence of this mutation leads to an increase in the average DA distance under all solution conditions except acidic pH, where shorter distances are observed for some of the polypeptide chains. With the Pro30 mutation, both pairs lose populations with DA distances < 20 A. The expansion of the polypeptide in the vicinity of the Pro30 mutation may be attributable to the increased stiffness of the polypeptide backbone (segment length ~5 residues) and to the helix-disrupting property of Pro. From Ca chemical shift analysis, the slight bias (10%) for residual helical conformation in the N-terminal region is abolished in the A30P protein in solution [260].

The finding that single mutations in a-syn are linked to familial early-onset forms of PD points to a central role for the protein in the etiology of the disease. A crucial question is whether a conformationally altered (misfolded) protein is directly involved in the pathogenic mechanism. Our analysis of the FET kinetics emphasizes that a-syn is a highly disordered polymer at pH 7.4, pH 4.4, and in the presence of micelles. On average, the polypeptide is more extended than expected for a freely jointed polymer, and under some conditions the protein is substantially less flexible than a random coil. Nevertheless, it is likely that the protein is highly dynamic and that conformers interchange on very short ^s) time scales. We find that modifications of solution conditions and amino-acid sequence do not produce unique conformational changes; rather, these perturbations result in subtle redistributions of the structures comprising the protein ensemble.

It has been suggested that the death of dopaminergic neurons in Parkinson's disease is the ultimate result of a cascade of events involving inhibition of mito-chondrial complex I, a-syn aggregation, and proteosome dysfunction [253,254]. The role of a-syn in this complex disease progression is likely to involve its interactions with other biomolecules (e.g., vesicles, enzymes, chaperones, pro-teosomes) [253,254]. FET kinetics measurements have provided unique insights into the conformational heterogeneity of a-syn that are not apparent from other spectroscopic measurements. The greatest power of this approach may lie in its ability to determine how different a-syn subpopulations interact with neuronal compounds and structures that are implicated in the pathogenic process.

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