synuclein fission yeast model - Springer Link

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Journal of Molecular Neuroscience Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN0895-8696/06/28:179–192/$30.00 JMN (Online)ISSN 1559-1166 DOI 10.1385/JMN/28:02:179

ORIGINAL ARTICLE

α-Synuclein Fission Yeast Model Concentration-Dependent Aggregation Without Plasma Membrane Localization or Toxicity

Katrina A. Brandis, Isaac F. Holmes, Samantha J. England, Nijee Sharma, Lokesh Kukreja, and Shubhik K. DebBurman* Biology Department, Lake Forest College, Lake Forest, IL 60045 Received July 1, 2005; Accepted September 7, 2005

Abstract Despite fission yeast’s history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because α-synuclein misfolding and aggregation are linked to Parkinson’s disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates α-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type α-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for α-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T α-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T α-synuclein formed aggregates faster than wild-type α-synuclein and at a lower α-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T α-synuclein did not target to the plasma membrane in fission yeast, not even at low α-synuclein concentrations or as a precursor step to forming aggregates. Despite α-synuclein’s extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that α-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of α-synuclein biology, and both organisms shed insight into α-synuclein’s role in PD pathogenesis. DOI 10.1385/JMN/28:02:179 Index Entries: α-Synuclein; Parkinson’s disease; neurodegeneration; Schizosaccharomyces pombe; protein aggregation; nucleation polymerization.

Introduction Parkinson’s disease (PD) is an incurable and fatal neurodegenerative disease, characterized pathologically by the death of midbrain substantia nigra neurons in the human brain (Olanow and Tatton, 1999; Dawson and Dawson, 2003). These dopamineproducing cells accumulate misfolded and aggregated α-synuclein in cytoplasmic inclusions called

Lewy bodies, which is linked to toxicity (Spillantini et al., 1998). Whether the amyloid fibril structures of aggregated α-synuclein found in Lewy bodies or the more soluble oligomers (protofibrils) of the protein are the true toxic culprits is still much debated (Lansbury, 1999; Goldberg and Lansbury. 2000; Haas and Steiner, 2001; Hardy and Selkoe, 2002; Taylor et al., 2002; Caughey and Lansbury, 2003). Several model

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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180 systems in mice (Dauer and Przedborski, 2003), worms (Lasko et al., 2003), flies (Feany and Bender, 2000; Auluck et al., 2002), and budding yeast (Outeiro and Muchowski, 2004) are being employed to better understand α-synuclein biology and the molecular mechanisms underlying the misfolding, aggregation, and toxicity of α-synuclein, as well as to clarify the α-synuclein species that causes toxicity. The nucleation polymerization hypothesis is the main model proposed to explain not only how misfolded α-synuclein aggregates into the amyloid fibrils seen in Lewy bodies but also the aggregation mechanism of other proteins linked to human neurodegenerative diseases, including Aβ, huntingtin, and the prion protein (Jarret and Lansbury, 1993; Eigen, 1996; Harper and Lansbury, 1997; Perutz and Windle, 2001; Caughey and Lansbury, 2003). This hypothesis states that soluble protein monomers aggregate into polymers over time through a process facilitated by the formation of an initial nucleus (a small oligomer) that seeds the polymerization process. Increased protein concentration or mole cular crowding facilitates the rate-limiting nucleus formation step. Several in vitro studies with wildtype and familial mutant (A30P and A53T) forms of purified recombinant α-synuclein support this model, yet cellular evidence has been sparse (Conway et al., 1998, 2000; Giasson et al., 1999; Nahri et al., 1999; Wood et al., 1999; Li et al., 2001; Shtilerman et al., 2002). Recently, however, in budding yeast, α-synuclein was found to aggregate within cells when expressed from two chromosomally integrated gene copies but remains exclusively plasma membrane associated when expressed from one chromosomally integrated gene copy (Outeiro and Lindquist, 2003). Saccharomyces cerevisiae (budding yeast) has emerged as a powerful genetic system to model protein misfolding-linked neurodegenerative diseases (Outeiro and Muchowski, 2004), including prion diseases (Ma and Lindquist, 1999), Huntington’s disease (Krobitsch and Lindquist, 2000; Muchowski et al., 2002), and amyotrophic lateral sclerosis (Kunst et al., 1997). In four budding yeast models used for studying α-synuclein properties, it was found that wild-type and A53T mutant α-synuclein are plasma membrane-associated proteins that can aggregate within cells; whereas the A30P mutant neither localizes to the plasma membrane nor aggregates (Outeiro and Lindquist, 2003; Dixon et al., 2005; Zabrocki et al., 2005; Sharma et al., 2006). However, the support for α-synuclein toxicity to budding yeast


Brandis et al. is varied: Two models demonstrate that α-synuclein aggregation itself causes toxicity (Outeiro and Lindquist, 2003; Dixon et al., 2005), whereas the other two report that α-synuclein-dependent toxicity requires an additional chemical or genetic insult, such as proteasomal inhibition and oxidative stress (Zabrocki et al., 2005; Sharma et al., 2006) or coexpression of the microtubule-associated protein tau (Zabrocki et al., 2005). Similarly, inhibition of phospholipid metabolism (Outeiro and Lindquist, 2003) and secretory pathway dysfunction (Dixon et al., 2005) can increase α-synuclein-dependent toxicity. Moreover, overexpression of α-synuclein in yeast can lead to abnormal lipid accumulation (Outeiro and Lindquist, 2003) and disrupt endocytosis (Outeiro and Lindquist, 2003; Zabrocki et al., 2005). Willingham et al. (2003) reported a budding yeast genetic screen that identified 86 genes that were synthetically lethal when absent with overexpressed α-synuclein, further underscoring the broad benefits of harnessing yeast as a model organism. Schizosaccharomyces pombe (fission yeast) is also a powerful model organism in cell biology, providing insight into the eukaryotic cell cycle (Fantes and Beggs, 2000), DNA repair and recombination (Davis and Smith, 2001), and checkpoint controls needed for stability (Humphrey, 2000). Like budding yeast, fission yeast shares with humans a high conservation of protein folding and protein quality control pathways (Wood et al., 2002), yet it has not been used to model the molecular bases of protein misfolding diseases, like PD. Therefore, we evaluated the potential of fission yeast to model α-synuclein pro perties and gain new insights, and we compared α-synuclein characteristics between S. pombe and S. cerevisiae. First, we asked if wild-type α-synuclein, familial mutants (A30P and A53T), and a double mutant (A30P/A53T) would misfold and aggregate in fission yeast. Second, we took advantage of thiaminerepressible pNMT vectors (pNMT81, pNMT41, and pNMT1) to express α-synuclein in increasing concentrations and tested the nucleation polymerization hypothesis for α-synuclein misfolding and aggregation directly in living cells. Third, we asked if nucleation-dependent α-synuclein aggregation (provided it does occur in fission yeast) led to concentration-dependent cytotoxicity. Finally, we asked if α-synuclein preserved plasma membrane-binding ability in fission yeast, as reported in budding yeast, and if this property was related to toxicity.

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Materials and Methods

Fluorescence Microscopy

S. pombe Expression Vectors Human wild-type and A53T mutant α-synuclein cDNAs were a gift from Christopher Ross (Johns Hopkins University). A30P and A30P/A53T mutant α-synuclein were created from wild-type and A53T mutant α-synuclein, respectively, using site-directed mutagenesis (Invitrogen). Polymerase chain reaction (PCR) was used to amplify C-terminal green fluorescent protein (GFP)-tagged α-synuclein (wild-type, A30P, A53T, A30P/A53T) fusion cDNA from the α-synuclein-GFP containing pYES2/TOPO S. cerevisiae vectors constructed by Sharma et al. (2006): forward primer, 5′-GGGGCCAAGCTTGC CATGGATGTATTCATGAAAGGA-3′; reverse primer, 5′-TTTGTAGAGCTCATACATGCCATG-3′. Similarly, PCR was used to amplify GFP cDNA from GFP-pYES2/TOPO S. cerevisiae vectors constructed by Sharma et al. (2006): forward primer, 5′CCCGGGACCATGGCCAGCAAAGGAGAAG-3′; reverse primer, 5′-TTTGTAGAGCTCATACATGC CATG-3′. These PCR products were subcloned, according to the manufacturer’s protocol (Invitrogen), into each of these three fission yeast pNMT TOPO-TA expression vectors: pNMT1 (for high expression), pNMT41 (for intermediate expression), and pNMT81 (for low expression) vectors. Note that these vectors added a V5epitope and a 6X histidine epitope at the C-terminal end of the subcloned α-synuclein-GFP sequence. These vectors were transformed into Escherichia coli, again according to the manufacturer’s protocol (Invitrogen). Positive transformants were verified for correctly oriented subcloned cDNA by standard bacterial wholecell PCR. Plasmid vectors were purified using a Qiagen miniprep kit, and the respective subcloned DNA sequences were confirmed (University of Chicago sequencing facility). The parent pNMT1, pNMT41, and pNMT81 pREP vectors were kindly provided by Judy Potashkin (Rosalind Franklin University of Medicine and Science, North Chicago, IL).

S. pombe cells were grown overnight at 30°C in Edinburgh minimal medium (EMM [Invitrogen]) containing thiamine (10 µM [to repress α-synuclein expression]). After 24 h, cells were pelleted at 1500g for 5 min, washed twice in 10 mL dH2O, resuspended in 10 mL EMM without thiamine, of which 125 µL cells were used to inoculate 25 mL EMM without thiamine (to express α-synuclein). Cells were prepared at desired expression time points for microscopy, as described in Sharma et al. (2006), and viewed using a Nikon TE-2000U fluorescence microscope at ×1000 magnification. Images were deconvoluted using MetaMorph version 4.2 software. Live-cell DAPI staining (for Fig. 1, below) was conducted using a procedure published in Alfa et al. (1993). For Fig. 4 (below), to quantify α-synuclein aggregates, cells were first viewed under differential interference contrast (DIC) microscopy, and total cell count in the field was determined and viewed for GFP fluorescence. The number of cells in the field containing 1, 2, or 3+ aggregates was determined. The field was then moved three turns on the field control knob, and the process was repeated in a new field. At least 750 cells were evaluated for each treatment. Aggregates were scored as percent of total cells in the field that expressed 1, 2, and 3+ aggregates.

Yeast Strains TCP1 (h– leu1-32; Invitrogen); SP3 (h+ leu1-32; kindly provided by Judy Potashkin, Rosalind Franklin University of Medicine and Science).

Yeast Transformation S. pombe strains were transformed with pNMT vectors using the lithium-acetate transformation method (Alfa et al., 1993).


Protein Expression Lysates of S. pombe expressing α-synuclein, protein electrophoresis, and Western blotting were performed as reported for S. cerevisiae in Sharma et al. (2006). The monoclonal anti-V5 antibody-AP (Invitrogen [1:5000 dilution]) was used for protein detection.

Growth Curve Transformed cells were grown in 10 mL EMM + thiamine overnight at 30°C (200 rpm). Cells were harvested at 1500g for 5 min at 4°C, washed twice in 5 mLdH2O, resuspended in 5 mLdH2O, and counted. Flasks with 25 mL EMM + thiamine or EMM – thiamine were each inoculated to 2.0 × 106 cells/mL density. At 0, 3, 6, 12, 18, 24, and 36 h, OD600 absorbance of 1 mLcell culture (in duplicate) was measured using a Hitachi U-2000 spectrophotometer. Averaged absorbance readings were plotted against time points.

Serial Spotting For spotting, transformed cells were grown to mid-log phase in EMM + thiamine, normalized to

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Fig. 1. GFP microscopy of α-synuclein in fission yeast. (A) Top panel: Cellular localization of wild-type, A30P, A53T, and A30P/A53T α-synuclein expressed from the pNMT1 vector at 18 h of growth in EMM without thiamine. Bottom panel: Magnified images of representative cells to highlight variable number of α-synuclein aggregates (one to five per cell) in wild-type and A53T cells, and diffuse fluorescence in A30P or A30P/A53T cells. (B) Controls: Cells containing parent pNMT1 vector exhibited no GFP fluorescence, nor did cells containing wild-type α-synuclein-GFP NMT1 vectors grown in thiamine, whereas GFP alone was cytoplasmically diffuse. (C) Analysis of the same cells with DAPI staining and GFP fluorescence confirms that α-synuclein aggregates are not localized within the nucleus. (D) α-Synuclein-GFP fluorescence patterns in a second fission yeast strain (SP3) were similar to TCP1.

equal densities (2 × 107 cells/mL), serially diluted (5-fold) into 96-well microtiter plates, and spotted on EMM + thiamine or EMM – thiamine plates using a 48-prong frogger (Dan-Kar) or manual pipeting. Images were scanned with a Bio-Rad Versadoc multi-imager after 2 d of growth.


Results Wild-type α-synuclein and its three mutant forms (A30P, A53T, A30P/A53T) tagged with GFP at the carboxyl terminus were expressed in the fission yeast strain TCP1, driven by the high expressing pNMT1

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α-Synuclein Aggregation in Fission Yeast vector. Cells were grown in EMM in the absence of thiamine to induce maximal protein expression. At 18 h of growth, live-cell GFP imaging revealed that wild-type and A53T α-synuclein-GFP coalesced into bright foci in almost all cells that expressed α-synuclein, indicative of the aggregation reminiscent of Lewy bodies in human substantia nigra neurons seen in PD (Fig. 1A, upper panel). These cells typically exhibited between one and five aggregates (a few cells contained more than five), but the most common number was one or two aggregates per cell (Fig. 1A, lower panel [representative cells magnified]). The A30P mutant, on the other hand, remained cytoplasmically diffuse, recapitulating its known resistance to aggregate into large inclusions (Fig. 1A). Interestingly, the A30P/A53T double mutant distributed very much like the A30P mutant. As expected, GFP expressed alone was cytoplasmically diffuse, whereas parent vector control and α-synucleinGFP-transformed cells grown in thiamine (which represses α-synuclein expression) showed insignificant fluorescence (Fig. 1B). Wild-type and A53T α-synuclein aggregates were cytoplasmic in location because they did not overlap with DAPI-stained nuclei (Fig. 1C). Finally, to confirm that this pattern of α-synuclein aggregation was not specific to just one fission yeast strain, we transformed α-synuclein into a second strain, SP3, and obtained strikingly similar fluorescence patterns for all four forms of α-synuclein (Fig. 1D). Given that wild-type and A53T α-synuclein aggregated in fission yeast, we next evaluated if the nucleation-polymerization model held true for this aggregation. First, we assessed time dependence by evaluating α-synuclein aggregate formation in high expressing pNMT1 cells over a 36-h time course in EMM without thiamine (Fig. 2A). At 6 h, neither wild-type nor mutant α-synuclein could be detected by GFP imaging, but wild-type α-synuclein began forming aggregates by 18 h. The A53T mutant began aggregating significantly earlier at 12 h. As expected, A30P and A30P/A53T α-synuclein remained cytoplasmically diffuse throughout the time course. To evaluate the concentration dependence, we conducted two experiments. First, we predicted that a 36-h time course performed with wild-type and mutant α-synuclein expressed in low levels with the pNMT81 vector would yield few to no α-synuclein aggregates, as α-synuclein concentration would be too low to form many nuclei to seed polymerization. That was the case, as only a few cells had α-synuclein aggregates at 24 or 36 h (Fig. 2B). Second, we grew


183 high expressing pNMT1 cells containing all four α-synuclein forms in increasing concentrations of thiamine (0.1, 1, and 10 µM), given that thiamine represses the NMT1 promoter and lowers α-synuclein expression in a dose-dependent manner (Western blot not shown). Wild-type and A53T α-synuclein aggregation was absolutely dependent on α-synuclein concentration: Neither aggregated at 10 µM thiamine, but both aggregated in its absence (Fig. 3). The A30P and A30P/A53T mutants did not aggregate in any of the given thiamine concentrations. Interestingly, wildtype aggregation started at 0.1 µM thiamine, whereas A53T aggregation started at 1 µM, suggesting that it needed lesser α-synuclein concentration to seed polymerization. As summarized in Fig. 4, using pNMT1 cells, we quantified the time and concentration dependence of aggregate formation for wild-type and A53T α-synuclein, by counting the number of yeast cells that contained 1, 2, and 3+ aggregates of α-synuclein, over the 36-h time course and under varying concentrations of thiamine (0, 0.1, 1, and 10 µM). Our results unambiguously confirm that (1) aggregation of α-synuclein is concentration-dependent and timedependent; (2) A53T aggregates more quickly and at lower α-synuclein concentration compared with wild-type α-synuclein; and (3) A30P never aggregates and is dominant over the A53T mutation, when both mutations (A30P/A53T) are present on the same protein. In all budding yeast models for α-synuclein, plasma membrane localization of the wild-type and A53T mutant is the most consistent property reported to date (Outeiro and Lindquist, 2003; Dixon et al., 2005; Zabrocki et al., 2005; Sharma et al., 2006). Surprisingly, we could not find one fission yeast cell among the thousands of cells examined expressing wild-type, A30P, A53T, or A30P/A53T α-synuclein from the pNMT1 vector that exhibited significant plasma membrane localization when examined between 18 and 24 h of growth (Fig. 1). However, the inability of the A30P mutant to bind the plasma membrane was not unexpected, as it cannot bind phospholipids in vitro and in cellular membranes (Jo et al., 2002; Bussell and Elizier, 2004). In budding yeast, α-synuclein can relocate from the plasma membrane into intracellular aggregates when cellular expression is doubled (Outeiro and Lindquist, 2003), or over extended time as α-synuclein accumulates in cells (Zabrocki et al., 2005). But lowering α-synuclein expression in fission yeast, either by using pNMT81 cells (Fig. 2B) or pNMT1 cells

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Fig. 2. Time course of α-synuclein aggregation. α-Synuclein was expressed using pNMT1 (A) or pNMT81 (B) vectors by growth in EMM without thiamine. Images were captured at the indicated times over 36 h. (A) Wild-type and A53T α-synuclein began to form aggregates at 18 and 12 h, respectively. A30P and A30P/A53T exhibited diffuse cytoplasmic fluorescence throughout the time course. (B) All forms of α-synuclein were diffuse in the cytoplasm throughout this time course, except for a few wild-type and A53T cells at 24 and 36 h.


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Fig. 3. Concentration dependence of α-synuclein aggregation. pNMT1 cells were grown in media in decreasing concentrations of thiamine (10, 1, and 0.1 µM) to produce increasing amounts of all four forms of α-synuclein. Images were captured at 24 h of growth. Wild-type and A53T α-synuclein began to form aggregates at 0.1 and 1 µM thiamine concentrations, respectively, but not at 10 µM thiamine. A30P and A30P/A53T α-synuclein were diffuse in the cytoplasm at all thiamine concentrations.

supplemented with thiamine (Fig. 3), did not yield membrane localization of α-synuclein. Furthermore, in fission yeast, α-synuclein membrane localization was not an obligatory initial event prior to aggregate formation, because nowhere in the time course of wild-type and A53T α-synuclein expression did pNMT1 cells exhibit membrane localization (Fig. 3). Cells that lacked α-synuclein aggregates instead exhibited cytoplasmically diffuse fluorescence. Finally, we asked if nucleation-dependent aggregation of α-synuclein led to cellular toxicity. In all experiments conducted in this study, cells were grown under stringent conditions (EMM) to maximize the possibility of uncovering toxicity. We performed OD600 growth curves of both GFP-tagged and nonGFP-tagged α-synuclein (Fig. 5), under conditions where α-synuclein expression was low (pNMT81 and/or pNMT41 cells) or high (pNMT1 cells). To our surprise, despite evidence for extensive aggregation


for wild-type and A53T α-synuclein in pNMT1 cells, these proteins were not toxic to yeast, nor were the A30P and A30P/A53T mutants (Fig. 5A). pNMT81 cells grew at similar rates compared with pNMT1 cells, irrespective of α-synuclein expression (–thiamine) or repression (+thiamine or parent vector) and irrespective of wild-type or mutant forms of α-synuclein. This nontoxicity was further confirmed by evaluating growth of α-synuclein-expressing pNMT81, pNMT41, and pNMT1 cells by serially diluting and spotting them onto EMM plates, with and without thiamine. No difference in growth was observed among cells that contained parent plasmid and those that expressed wild-type or mutant forms of α-synuclein, either at low or high levels (Fig. 5B). Western blotting confirmed that untagged α-synuclein was expressed at increasing levels, from pNMT81, pNMT41, and pNMT1 vectors, respectively (Fig. 5C [similar data obtained with GFP-tagged α-synuclein

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Fig. 4. Summary of quantified wild-type and A53T α-synuclein aggregation. Cells expressing wild-type and A53T α-synuclein over a 36-h time course with decreasing concentrations of thiamine (0.1, 1, and 10 µM) were counted for the number of aggregates formed per cell, as described in Materials and Methods. Cells were scored as containing 1 aggregate (light gray bar), 2 aggregates (white bar), or 3+ aggregates (black bar). Bars represent percentage of 750–1000 total cells counted in each sample that exhibited the designated number of aggregates per cell.

are not shown]). Interestingly, similar to observations in our budding yeast model (Sharma et al., 2006), both untagged and GFP-tagged α-synuclein resolved higher than expected on protein gels: Untagged α-synuclein migrated at ~24–26 kDa instead of the predicted 18 kDa (includes 3- to 4-kDa V5/6X his tag [Fig. 5C]), whereas GFP-tagged α-synuclein ran at ~52–54 kDa instead of the predicted 45 kDa (data not shown).

Discussion Using the PD-linked protein α-synuclein as a prototype, we have described the first fission yeast model to study the misfolding and aggregation prop-


erties of proteins linked to human neurodegenerative diseases. In addition to recapitulating α-synuclein’s normal and pathological characteristics, the model illustrates a notable difference in α-synuclein property between fission and budding yeast models for α-synuclein, that is, its ability to associate with the plasma membrane. First, live-cell evidence strongly supports that a nucleation-polymerization model for α-synuclein aggregation occurs in vivo. This model has widespread acceptance for α-synuclein (Conway et al., 1998, 2000; Giasson et al., 1999; Nahri et al., 1999; Wood et al., 1999; Li et al., 2001; Shtilerman et al., 2002) and other toxic proteins in neurodegenerative diseases

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Fig. 5. Toxicity and expression analysis of α-synuclein. (A) Growth curve: OD600 measurements over 24 h were obtained for cells containing wild-type, A30P, A53T, and A30/A53T forms of α-synuclein that were tagged with GFP (in pNMT1 and pNMT81 vectors), as well as those that were untagged (in pNMT1, pNMT41, and pNMT81 vectors). All cultures were grown either without (solid lines) or with (broken lines) thiamine. Concentration-dependent α-synuclein toxicity to fission yeast was not observed, irrespective of whether α-synuclein was tagged with GFP or not. (B) Spotting: Serially diluted cells expressing all four forms of untagged α-synuclein in pNMT1, pNMT41, and pNMT81 vectors or containing parent vectors were spotted onto EMM plates without (–T) or with (+T) thiamine. No noticeable differences in growth were observed between cells expressing α-synuclein and those expressing the parent plasmid after 2 d of growth. (C) Western analysis: Cells containing untagged α-synuclein in pNMT1, pNMT41, and pNMT81 vectors were grown in the presence (+) and absence (–) of thiamine for 18 h. Cells containing pNMT1 vectors showed strongest protein expression, whereas expression in pNMT81 cells was negligible, except for wild-type α-synuclein. Addition of thiamine either reduced protein expression (pNMT1) or completely repressed α-synuclein expression (pNMT41 and pNMT81).

(Eigen, 1996; Perutz and Windle, 2001; Caughey and Lansbury, 2003), yet live cell and in vivo support have only recently been forthcoming. In fission yeast, wildtype and A53T α-synuclein require high cellular expression to form intracellular inclusions. A nucleation-like event facilitated by increased αsynuclein concentration likely occurs in these living cells, best explaining its aggregation pattern. However, unlike that suggested in some budding yeast models (Outeiro and Lindquist, 2003; Zabrocki et al., 2005) and in mammalian cell membranes (H. J. Lee et al., 2002), α-synuclein aggregation does not require initial membrane localization in fission yeast. Second, both α-synuclein familial mutants A30P and A53T recapitulate in fission yeast well-known


properties reported previously in vitro and in mammalian cell model systems (Conway et al., 1998, 2000; Giasson et al., 1999; Nahri et al., 1999; Wood et al., 1999). Specifically, the A53T mutant aggregates into inclusions within live fission yeast and is more prone to aggregation compared with wild-type α-synuclein, whereas the A30P mutant does not form inclusions. The A30P mutant dominates over A53T in the double mutant A30P/A53T, as reported by Jo et al. (2002). But while A30P prevents membrane binding of the double mutant in the earlier study (Jo et al., 2002), we found that it prevented the formation of intracellular inclusions in this study. These findings strengthen the notion that A30P and A53T mutations cause disease via different mechanisms.

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188 Third, α-synuclein is nontoxic to fission yeast despite extensive aggregation and growth in stringent minimal conditions, suggesting that aggregated α-synuclein within inclusions in fission yeast is either inherently protective or harmless. Although still a controversial notion, studies with animal models of other neurodegenerative diseases have reported that protein inclusions can form without associated toxicity (Rochet et al., 2000) or that neurotoxicity can ensue without obvious protein inclusions (Sisodia, 1998; Caughey and Lansbury, 2003). In our model we speculate that if aggregates of α-synuclein do have toxic properties, then the yeast protects against it. As Willingham et al. (2003) uncovered 86 genes in budding yeast that conferred protection against α-synuclein toxicity, future genetic screens in S. pombe might lead to the discovery of similar or new genes relevant to PD neuropathology. The A30P mutant is neither toxic to fission yeast nor does it form inclusions. This mutant is linked to neuronal cell death in PD and in animal models (Dawson and Dawson, 2003), and forms cytotoxic protofibrils with a reduced ability to polymerize into amyloid fibers (Conway et al., 1998, 2000; Li et al., 2001). In fission yeast the A30P mutant either does not accumulate toxic protofibrils, or the yeast protects against it if it does. Fourth, α-synuclein exhibits three properties differently in budding and fission yeast.

Property 1 Wild-type and A53T α-synuclein do not localize to the fission yeast plasma membrane, contrary to mounting evidence that α-synuclein is a lipidbinding protein in vitro (Jensen et al., 1998; Cole et al., 2000; Perrin et al., 2000, 2001; Sharon et al., 2001; Lotharius and Brundin, 2002; Necula et al., 2003) and binds the plasma membrane in budding yeast (Outeiro and Lindquist, 2003; Dixon et al., 2005; Zabrocki et al., 2005; Sharma et al., submitted). It should be noted, however, that in neuronal cells α-synuclein is mostly distributed in the cytosolic fraction, either in cell bodies or near presynaptic vesicles, with minor or transient localization at the plasma membrane (Maroteaux and Scheller, 1991; Shibayama-Imazu et al., 1993; George et al., 1995; Irizarry et al., 1996; Iwai et al., 1995; Kahle et al., 2000; McLean et al., 2000). We speculate that the lack of membrane association in fission yeast is attributable to reduced or altered plasma membrane phospholipid composition, or differences in membrane proteins with which α-synuclein interacts. Although not compared extensively, budding and fission yeasts have reported modest


Brandis et al. differences in phospholipid composition, particularly phosphatidylethanolamine (PE [Fer nandez et al., 1986]). Interestingly, α-synuclein inter acts with the phosphatidylinositol signaling pathway (Narayanan et al., 2005) and preferentially binds to acidic phospholipids in vitro (Davidson et al., 1998; Jo et al., 2000; Perrin et al., 2000), with PE increasing this interaction (Jo et al., 2000).

Property 2 Despite the lack of toxicity in fission yeast, when α-synuclein forms cytoplasmic inclusions in budding yeast, it can be strongly toxic (Outeiro and Lindquist, 2003), suggesting that the two yeasts either handle α-synuclein toxicity differently or produce differing levels of toxic protein. α-synuclein affinity for membranes increases as the protein forms protofibrils and reduces as it forms amyloid fibrils (Volles et al., 2001; Ding et al., 2002; Volles and Lansbury, 2002). Therefore, membrane localization might be necessary to generate enough toxic protofibrils (M. K. Lee et al., 2002; Rochet et al., 2004) and/or toxic amyloid pores (Lashuel et al., 2002), which might explain why α-synuclein is more toxic to budding yeast.

Property 3 In examining the A30P/A35T double mutant in fission yeast, the A30P mutation suppresses the aggregation-inducing property of the A53T mutation. However, in budding yeast (Sharma et al., 2006), A30P/A35T retains both aggregation- and membrane-associated properties, therefore, behaving more like wild-type and A53T rather than A30P. Thus, the cellular/organismal context influences the extent to which two mutations in α-synuclein dictate if the protein aggregates and/or binds lipids.

Conclusions In conclusion, fission yeast demonstrates nucleation-dependent α-synuclein misfolding and aggregation, the presence of cytoplasmic inclusions, and the inability of the A30P mutant to aggregate. Plasma membrane localization and lipid-binding ability of α-synuclein are cell-specific properties. Plasma membrane localization of α-synuclein might be a missing key step in fission yeast needed for toxicity. Future genetic screens and candidate gene approaches, as well as chemical treatments, will help dissect the role of genetic factors that regulate α-synuclein misfolding and toxicity in this organism and contribute to deeper understanding of PD pathogenesis.

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Acknowledgments At Lake Forest College, we thank Dr. Douglas Light and undergraduates, Sara Herrera, Tulaza Vaidya, Tasneem Saylawala, Mithaq Vahedi, and Sina Vahedi, for editorial comments. Thanks go to Dr. Judy Potashkin (Rosalind Franklin University of Medicine and Science) for encouraging us to develop the fission yeast model for α-synuclein and Dr. Virginia McDonough (Hope College, Holland, MI) for discussions on lipid physiology in yeasts. S. D. was supported by grants from NIH (R15 grant no. NS048508), NSF (CCLI 0310627) and MRI (0115919), Campbell Foundation (Grand Rapids, MI), Lake Forest College, and a MacArthur grant from Kalamazoo College. I. H. was supported by a 2004 Parkinson Disease Foundation summer undergraduate research fellowship, and N. S. was supported by a 2003 Council of Undergraduate Research fellowship.

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