Journal of Molecular Biology
Heat Shock Prevents Alpha-synuclein-induced Apoptosis in a Yeast Model of Parkinson's Disease
Introduction
Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The hallmarks of PD are the selective loss of the cells comprising the substantia nigra pars compacta and the presence of proteinacious inclusion bodies termed Lewy bodies and Lewy neurites in the affected neurons.1 The discovery that the protein α-syn, which is an intrinsically unfolded protein of unknown function,2, 3 is the main component of Lewy bodies has sparked an intense search for the mechanism of α-syn toxicity.4 Wild-type α-syn (WT) is associated with sporadic PD, whereas the two mutants, A53T and A30P,5, 6 are associated with autosomal dominant, early-onset PD. It has been shown that α-syn binds to lipids,7 inhibits phospholipase D,8 and is subject to complex association reactions leading to 10 nm fibers.9, 10, 11 Whether large fibers of α-syn or smaller protofibrils are the toxic agent is the subject of intense investigations. Model systems such as Drosophila melanogaster,12 Caenorhabditis elegans,13, 14 and the budding yeast, Saccharomyces cerevisiae,15, 16 are being used to uncover the mechanism of α-syn toxicity. Human molecular genetic studies continue to discover other genes (PINK-1, DJ-1, and LRRK-2) associated with early onset PD.17, 18, 19
α-syn accumulates in presynaptic vesicles where it is thought to regulate neurotransmitter release and contribute to synaptic plasticity.20, 21, 22 Other members of the synuclein family are β and γ-syn; however, only α-syn forms amyloid-like fibers. Synucleins display an unusual heptameric motif KTK(E/Q)GV that is repeated six times. Wild-type α-syn binds to negatively charged phospholipid membranes and this interaction promotes an unfolded to folded transition in the protein, resulting in the formation of two long α-helices that interact with the membrane.23 The helical regions of α-syn comprise residues 1–44 and 45–100. The A53T mutant also interacts with the plasma membrane, whereas there are conflicting reports as to whether the A30P mutant localizes to the plasma membrane.24, 25 Proline, which is a helix breaker, could disrupt the unfolded to folded helix transition in the N-terminal helix and thus prevent association with the plasma membrane, which might explain why a green fluorescent protein fusion of A30P localizes in the cytoplasm of yeast cells.15 All three α-syns are subject to complex fibrillization reactions. Protofibrils and fibrils disrupt membranes26 and impair proteasome function.27, 28 Both processes could lead to cell death.
Recent studies have shown that the accumulation of wild-type (WT) α-syn in cultured neurons induces apoptosis29, 30, 31 and that α-syn's toxicity is related to dopamine production.32 In one of these studies, Xu and colleagues32 discovered that expressing WT α-syn in human fetal dopaminergic neurons causes apoptosis, as indicated by chromatin condensation, nuclear fragmentation, and reactive oxygen species (ROS), whereas α-syn expression in non-dopaminergic human cortical neurons protects cells and significantly increases neuronal survival. In another study, it was shown that apoptosis occurs in human neuroblastoma cells (SH-SY5Y) due to mutations that impair the function of DJ-1.33
The objective of this study was to determine whether expression of α-syn in non-dopaminergic cells can induce apoptosis, and S. cerevisiae was chosen as a model because it is amenable to genetic analyses. Yeast has been shown to undergo an apoptotic-like cell death, e.g. loss of membrane asymmetry, cytochrome c release, and DNA fragmentation, as a consequence of a mutation in the AAA+ ATPase allele CDC48,34 expression of proapoptotic human genes (bax or p53),35, 36 exposure to H2O2,37 acetic acid,38 and other insults;39, 40 however, the precise mechanisms by which these different insults trigger apoptosis in yeast are not understood as well as for mammalian cells. Yeast contain two anti-apoptotic 14-3-3 orthologues (BMH1 and BMH2),41, 42 a pro-apoptotic metacaspase (YCA1),43 an apoptosis inducing factor orthologue (AIF1),44 and a DJ-1 orthologue,45 which reinforces the relevance of using yeast to understand the pathobiology of α-syn. Orthologues of yeast genes that protect S. cerevisiae from α-syn may also protect human cells.
Section snippets
Results
The budding yeast S. cerevisiae was used in the experiments described here. The genotypes of the various yeast strains and the plasmids used are given in Table 1, Table 2, respectively. In addition to analyzing how expression of the human α-syn transgene affects yeast cells, parallel experiments were conducted on yeast cells expressing human β-syn, which is a close relative of α-syn. Unlike α-syn, β-syn produces no motor defects when it is expressed in the brains of mice.46 Interestingly,
Discussion
This study has shown that human α-syn (WT, A53T, or A30P) triggers apoptosis in S. cerevisiae, and that the ability of these three synucleins to induce apoptosis is exquisitely specific in that, β-syn, which is 62% identical with WT, does not induce apoptosis. Although α-syn induces apoptosis in yeast, the effects of α-syn in the nervous system are thought to be degenerative, thus it is not clear at present how the effects in yeast relate to neuro-degenerative changes. On the other hand, using
Strains, plasmids, and media
The primary yeast strain used in this study was FY23 (Table 1).69 FY23 cells were typically pre-grown in synthetic sucrose (2% w/v) medium lacking tryptophan (SSuc–Trp) to maintain selection. Medium containing synthetic sucrose is referred to as non-inducing medium. α-syn expression was induced in the same medium with galactose (2% w/v) replacing sucrose (SGal–Trp).70 Medium containing synthetic galactose is referred to as inducing medium. Several experiments were conducted with yeast strains
Acknowledgements
This work was funded, in part, by a grant from the Parkinson's Resource of Northwest Louisiana. We thank Neal Mathias for providing us with the yeast plasmids harboring the gene coding for the various GFP-α-syn fusions; Roland Lill and Lucy Robinson for antibodies; Peter Lansbury, Michael Volles, and John Kessler for providing us with the β-syn cDNA; Tony Fink for the pRK172 plasmid; Debra Chervenak for her assistance with FACS; Kelly Tatchell for use of his fluorescence microscope; and Tony
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