Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of cytoplasmic Lewy bodies in surviving neurons. The primary fibrillar component of Lewy bodies is α-synuclein. Two mutations have been linked to an early onset form of the disease, A30P and A53T. These neuropathological and genetic data suggest a link between PD and α-synuclein aggregation.

α-Synuclein is an abundant presynaptic protein. Although the physiological role of the protein has yet to be established, the primary sequence of α-synuclein suggests a membrane binding function. The N-terminal portion is comprised of seven 11-residue repeats with a highly conserved hexamer motif (KTK(E/G)V), similar to apolipoproteins. In solution, α-synuclein is natively unfolded (1); however upon binding membranes, it forms a helical structure (2, 3). Variants of α-synuclein demonstrate divergent membrane binding characteristics: A30P has lower affinity for synthetic and intracellular vesicles than A53T and wild-type (WT) (4, 5, 6, 7), most likely due to disruption of the helical structure described above (8). However, little is known about the influence of primary structure of synuclein variants on membrane binding. In addition, evidence suggests that α-synuclein membrane binding may seed the aggregation of the protein, suggesting a potential nucleation site for the misassembly of the protein.

In order to monitor the aggregation state of α-synuclein, we developed an assay using a strategy developed by those who study the yeast prion protein Sup35p. This assay was established such that the aggregation state of α-synuclein is reflected by a macroscopic property of the yeast. Sup35p demonstrates classical prion characteristics in that it can form aggregates, effectively leading to inactivation of the protein. The protein is an essential subunit of the eukaryotic release factor that upon binding Sup45p catalyzes the release of peptides from the ribosome when the ribosome encounters a stop codon. The prion state of Sup35p is investigated using a strain of Saccharomyces cerevisiae engineered to express the ade1-14 allele that encodes a nonsense codon in the coding region of the ADE1 gene encoding an essential enzyme in the biosynthetic pathway of adenine (9). When Sup35p is soluble, ([psi-]), yeast are unable to synthesize adenine due to truncation of the biosynthetic pathway. When Sup35p is insoluble ([PSI+], prion state), the protein is inactive and partial readthrough of the ADE1 gene product allows for growth on selective media lacking adenine (Figure 1, below).

Sup35p consists of three domains: N-terminal (the prion determinant), middle, and C-terminal domain. Since the N-terminal domain is required for aggregation of Sup35p, this domain was replaced with variants of α-synuclein to determine the effect of these α-synuclein-Sup35pMC fusion proteins on endogenous Sup35p in [psi-] yeast.

Preliminary experiments in these yeast demonstrated that while WT- and A53T-MC conferred a suppression phenotype, allowing for growth on selective media lacking adenine, A30P-MC did not. In parallel work, we and others have demonstrated that WT and A53T localize to the yeast plasma membrane while A30P is diffuse in the cytosol (10). In comparing the phenotypes of all synuclein variants investigated in the yeast model, we have found that the suppression phenotype tightly correlates with membrane binding affinity. These data suggest that we have developed a yeast model to monitor α-synuclein membrane binding. This model can be used in a highly efficient manner to screen for random mutants of α-synuclein with different propensities to bind membranes.

The model described above can also be used to investigate the effect of oxidation on α-synuclein lipid binding. Oxidative stress appears to play a key role in the pathogenesis of PD (11). This hypothesis was further confirmed by the identification of two mutations in the DJ-1 gene that lead to a loss of function phenotype and early onset PD (12). DJ-1 has antioxidant activity (13) and downregulation of the protein results in increased sensitivity to H₂O₂-induced neuronal cell death (14), suggesting that PD associated with these mutations is a result of increased oxidative stress. There is little information on how post-translation modifications such as methionine oxidation affect α-synuclein membrane binding. We have created a methionine sulphoxide reductase (MsrA) knock-out of the [psi-] yeast in order to determine the effect of methionine oxidation on α-synuclein membrane binding in our yeast model. Preliminary results suggest that methionine oxidation in this model may inhibit membrane binding.

In addition to the yeast model, we are also investigating the effect of DJ-1 on α-synuclein mediated neurotoxicity in a cellular model of PD. Several groups have demonstrated that overexpression of A53T α-synuclein in primary mesencephalic neurons results in selective toxicity to dopaminergic neurons (15). We hypothesize that co-overexpression of DJ-1 in this model of PD will alleviate the neurotoxicity of A53T. We are currently testing this hypothesis by simultaneously transducing rat primary neurons with A53T and DJ-1 lentivirus to determine the effect of DJ-1 on A53T toxicity. Conversely, we are also using siRNA to determine the effect of DJ-1 downregulation on primary neuron survival.

The research described above will contribute greatly to the field of PD research in that it will define a concise role for α-synuclein membrane binding in the pathogenesis of the disease. In addition, these studies will further elucidate the importance of oxidative stress and it's effect on α-synuclein toxicity, thus identifying potential drug targets for the treatment, prevention or cure of the disease.

Figure 1. [PSI+] and [psi-] phenotypes in S. cerevisiae encoding the ade1-14 allele. The ade1-14 allele encodes a premature stop codon in the ADE1 gene. In [psi-] yeast (A), Sup35p is soluble and active as a translation termination factor. The translation of the ADE1 gene is prematurely arrested and the yeast lack the ability to synthesize adenine. These yeast do not grow on selective media lacking adenine. In [PSI+] yeast (B), Sup35p is insoluble (prion state) due to extensive interactions at the N-terminal domain, translation termination is suppressed and partial readthrough of the ADE1 gene occurs. (Back to article.)

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