5 December 2003. The accumulation of protein aggregates is a common theme uniting most major neurodegenerative diseases (see related ARF Live Discussion), but does this phenomenon underscore a common pathogenic mechanism? While many scientists believe this assumption is correct, new data in today’s Science appears to throw some cold water on it. In back-to-back papers, Susan Lindquist and colleagues at MIT, collaborating with Paul Muchowski and colleagues at the University of Washington, Seattle, describe experiments with yeast as a model system to probe the toxicity of α-synuclein and huntingtin, proteins known to cause Parkinson's (PD) and Huntington's diseases (HD), respectively.
In the first paper, Lindquist and first author Tiago Fleming Outeiro focus on the properties of α-synuclein. In Parkinson's disease, mutants of this protein end up in cytoplasmic inclusions called Lewy bodies, suggesting that there may be a breakdown of the normal mechanisms that either keep the protein folded correctly (see ARF related news story) or remove unwanted synuclein for recycling (see ARF related news story). Because these mechanisms are biologically conserved among the eukaryotes, yeast provides a simple and pliable tool to test these theories, as well as to learn more about the function of α-synuclein, which is still poorly understood.
Fleming Outeiro used green fluorescent protein chimeras to track the cellular location of wild-type and mutant forms of α-synuclein in Saccharomyces cerevisiae. When the authors introduced single-gene copies of normal protein or the human A53T mutant, both localized to the plasma membrane, but not to mitochondrial or nuclear membranes. In contrast, the human A30P mutant was found to accumulate in the cytosol. To test the hypothesis whereby overpowered protein folding or recycling mechanisms contribute to α-synuclein toxicity, the authors increased its dosage to two or more genes. With two copies, even normal protein, and to a lesser extent the A53T mutant, were found to accumulate in large cytoplasmic inclusions. Significantly, the plasma membrane was virtually devoid of the chimeras under these conditions, indicating that lack of sufficient membrane binding sites was not the reason for the formation of protein aggregates. Staining cells for ubiquitin, the authors show that accumulation of synuclein is accompanied by accumulation of this peptide; however, as with human cells, not all synuclein aggregates stained positive for ubiquitin. Next, to test if the ubiquitin-proteasome degradation pathway is generally repressed in yeast expressing high copies of α-synuclein, the authors coexpressed an unstable GFP—GFPu—which is usually removed rapidly from the cell. All three variants of α-synuclein caused GFPu to accumulate, also, indicating a general suppression of the recycling machinery. Together, these lines of evidence support the theory that increasing the concentration of synuclein by modest amounts is sufficient to overwhelm the proteasome. The data also dovetails with the recent discovery that triplication of the synuclein gene in humans is sufficient to cause Parkinson’s disease (see ARF related news story).
The authors then turned their attention to the cellular function of α-synuclein. Prior work has suggested that the protein may regulate the trafficking of synaptic vesicles (see ARF related news story), and that it inhibits phospholipase D (PLD) in vitro. Fleming Outeiro and Lindquist show that wild-type and the A53T mutant of α-synuclein block the growth of yeast mutants that are sensitive to PLD inhibition (the A30P mutant had no effect). Further evidence for α-synuclein's role in lipid metabolism includes the observation that yeast expressing the Parkinson's protein accumulate lipid droplets, and that the distribution of the lipid-soluble fluorescent dye FM4-64 is dramatically altered by all three synuclein variants. Significantly, expression of toxic amounts of a polyglutamine-expanded fragment of huntingtin had no effect on the PLD-sensitive yeast cells, or yeast lipid metabolism, suggesting that protein aggregation per se may be but one aspect of synuclein toxicity.
A similar conclusion emerges from the second paper, in which first author Stephen Willingham, at University of Washington, and colleagues describe a genome-wide screen to identify proteins that exacerbate the toxicity of either α-synuclein or huntingtin. Willingham tested for genes that may keep the toxicity of these proteins at manageable levels by examining the viability of almost 5,000 different yeast strains, each devoid of one of two copies of a non-essential gene, and expressing either a polyglutamine-expanded fragment of huntingtin or α-synuclein.
Though evidence in the literature suggests there are similarities to huntingtin and synuclein toxicities, Willingham found only one mutant which was sensitive to expression of both proteins. This is in stark contrast to 86 mutants which were sensitive only to α-synuclein expression, and 52 mutants sensitive only to huntingtin expression, suggesting that the two proteins have widely disparate toxic properties.
This conclusion gains strength when Willingham lists the yeast deletion mutants whose functions are known. In the case of huntingtin, most of the 40 known proteins are involved in the stress response (20 percent), protein folding (7.5 percent), or ubiquitin-dependent protein recycling (7.5 percent). By contrast, of the 57 known proteins that exacerbate synuclein toxicity, most are involved in vesicle-mediated transport (18 percent), lipid metabolism, and transport (14 percent each). "Collectively," the authors state, "these results suggest that distinct pathogenic mechanisms may underlie HD and PD." The one yeast mutant that exacerbated both toxicities was for the transcription factor STP2.
As many of the yeast genes identified in this screen have human orthologs, this study may potentially lead to the discovery of new pathways that impact neurodegenerative diseases.—Tom Fagan.
Outeiro TF, Lindquist S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 2003 Dec 5;302(5651):1772-5.
Willingham S, Fleming Outeiro T, DeVit MJ, Lindquist SL, Muchowski PJ. Yeast genes that enhance the toxicity of a mutant huntingtin fragment or α-synuclein. Science. 2003 Dec 5;302(5651):1769-72.