A hydrogen peroxide-based genetic screen identifies suppressors of WT α-syn toxicity
We previously reported, using a high copy yeast genomic library to find suppressors of the super sensitivity of A30P, α-syn expressing cells to killing by hydrogen peroxide (22
). This same screen was used herein to identify suppressors of the super sensitivity of WT α-syn expressing cells to killing by hydrogen peroxide. The basis of the screen is that α-syn (WT, A30P or A53T) causes oxidative stress in a variety of cells (14
), including yeast (15
), which in turn results in the accumulation of ROS in the cytoplasm. Because cells expressing α-syn (WT, A30P or A53T) are burdened with endogenous oxidants, they cannot tolerate exogenous oxidants. To exploit this phenomenon, cells harboring pTF201 (WT α-syn) and a high copy yeast library were pre-grown in non-inducing media until mid-log phase and then shifted into inducing media and plated on solid agar plates that contained a disk of concentrated hydrogen peroxide. Such cells exhibited a sizeable halo of inhibition (~3 cm diameter) surrounding the peroxide disk where colonies failed to grow (Fig. A). In contrast, cells harboring pTF201 (WT α-syn) and the library often exhibited colonies within this halo of inhibition (Fig. B). Library plasmids from such colonies were isolated and sequenced as described in the Materials and Methods section. Strains and plasmids used in this study are given in Table .
Figure 1. A genome-wide screen identifies suppressors of the super sensitivity of WT α-syn expressing cells to killing by hydrogen peroxide. (A) FY23 cells transformed with pTF201 (WT α-syn) were plated on SGal−Trp. The halo of inhibition (more ...)
Using this approach, ~100 protective library plasmids were recovered. Because library plasmids contained yeast chromosomal fragments, some of the library plasmids could contain more than one gene. Therefore, both strands from each protective plasmid were sequenced, and with this sequence information the identities of genes on each plasmid were determined using the Saccharomyces
Genome Database (http://www.yeastgenome.org/
). Some recovered plasmids contained more than one gene. Instead of subcloning all of the genes from the recovered library plasmids, many of the genes were available in expression vectors from Open Biosystems. In total, we purchased sixty-six 2µ expression vectors (Open Biosystems), which contained the genes of interest behind the GAL1
promoter (Supplementary Material, Table S1
). To determine which genes conferred protection against WT α-syn toxicity, plasmids were introduced into the yeast strain FY23, and ROS accumulation was monitored using the dye dihydrorhodamine 123 (DHR123) (22
). DHR123 is a non-fluorescent cell permanent dye that fluoresces when oxidized. The basis of this secondary screen was that any gene that protects against WT α-syn toxicity should decrease the amount of cytoplasmic ROS and therefore decrease the amount of DHR fluorescence. Conversely, any gene that does not protect should not affect the DHR fluorescence.
The ability of the 66 genes, identified in the hydrogen peroxide screen, to inhibit ROS accumulation in cells expressing WT α-syn was determined. Cells were transformed with pTF201 (WT α-syn) and with a 2µ plasmid (BG1805) containing one of the protective genes. Transformants were pre-grown in non-inducing media to mid-log phase and then shifted into inducing media. After 2 h, the DHR123 dye was added and cells were incubated for an additional hour. The controls consisted of cells transformed with pTF201 (WT α-syn) or various control plasmids with no inserts. Fluorescence, which correlates with cytoplasmic ROS accumulation, was detected using a standard plate reader.
Figure shows the results from the ROS fluorescence assay. First, yeast cells expressing WT α-syn (+gal) yielded an ROS fluorescence signal of 1.5 (±0.2) × 105
units, whereas yeast cells with no WT α-syn expression (−gal) yielded an ~6-fold smaller ROS fluorescence signal of 2.7 (±0.5) × 104
units. Secondly, overexpression of 40 different genes decreased ROS accumulation by 30–60% (P
<< 0.05), compared with cells expressing WT α-syn only, and 24 of these have a human ortholog. These 24 protective yeast genes with human orthologs are given in Table . Thirdly, overexpression of several genes decreased ROS accumulation by 10–29%, compared with cells expressing WT α-syn only; however, we did not focus on these genes because of their relatively small effect. Fourthly, overexpression of nine genes had either no effect or slightly increased ROS accumulation, compared with cells expressing WT α-syn only. These nine genes were categorized as non-protective and were not evaluated any further. The complete list of genes and P
-values is given in Supplementary Material, Table S1
Figure 2. ROS fluorescence assay of transformants with 66 yeast genes identified in the hydrogen peroxide screen as potential suppressors of WT α-syn toxicity. The y-axis is the fluorescence signal; the x-axis gives the gene name. The FY23 strain was transformed (more ...)
Yeast genes with human orthologs that suppress WT α-syn toxicity
Deletion of protective genes enhances wt α-syn toxicity
were chosen for a more detailed analysis. These non-essential genes with human orthologs were chosen because four of them, ARG2
, conferred the greatest protection against WT α-syn-induced ROS, and because HSP82
encodes for a ubiquitous chaperone (Hsp90p) that, in yeast, is required for pheromone signaling and negative regulation of heat shock factor, Hsf1p (26
). Arg2p is a mitochondrial enzyme that catalyzes the first step in the biosynthesis of ornithine, which is a precursor of arginine (27
). Ent3p is involved in clathrin recruitment and protein transport between the trans
-Golgi network (TGN) and the vacuole (28
). Idp3p is a peroxisomal variant of the enzyme isocitrate dehydrogenase (29
). Jem1p is a DnaJ-like chaperone that localizes to the ER and is required for nuclear membrane fusion during mating (30
Given that these five genes in high copy suppressed the toxicity of WT α-syn, we reasoned that the deletion of any of them should enhance the toxicity of WT α-syn. Figure shows a viability assay in which the five haploid deletion strains and the parental wild-type haploid strain KT2253 were transformed with WT α-syn (pJL101) or a control plasmid with no insert (pJL100), serially diluted and spotted on sucrose and galactose plates. In all cases, without WT α-syn expression (−gal), growth of cells that contained the WT α-syn plasmid resembled that of cells containing the control plasmid. In contrast, the five haploid deletion strains (ent3Δ, idp3Δ, jem1Δ, arg2Δ and hsp82Δ) expressing WT α-syn grew much more slowly than the deletion strains harboring the control plasmid (+gal). The five haploid deletion strains expressing WT α-syn also grew much more slowly compared with the wild-type strain (KT2253) expressing WT α-syn (+gal). No appreciable difference in growth was observed for the wild-type strain (KT2253) expressing WT α-syn (+gal) versus the control plasmid with no insert (+gal), indicating that cells can tolerate one copy of WT α-syn. The combined results demonstrate that the deletion of these various genes enhances the toxicity of WT α-syn and suggests a specific protective function of the various gene products.
Figure 3. Growth properties of cells expressing WT α-synuclein. The effect of WT α-synuclein expression on the growth of five deletion strains (ent3Δ, idp3Δ, jem1Δ, arg2Δ and hsp82Δ) and the WT control strain (more ...)
The five deletion strains ent3Δ, idp3Δ, jem1Δ, arg2Δ and hsp82Δ expressing WT α-syn were also tested for ROS accumulation and for complementation of any ROS increase by expression of the corresponding gene. Similar to the experiment shown in Figure , cells were pre-grown in non-inducing media until mid-log phase and then shifted into inducing media. The ROS signal was measured after 3 h in inducing media. Each strain was transformed with pJL101 (WT α-syn) and a 2µ plasmid (Open Biosystems) with the gene of interest or pJL200 (empty 2µ control plasmid) (Table ).
Figure shows the results from the rescue experiments. First, the control strain KT2253 expressing WT α-syn (pJL101) and harboring a 2µ empty plasmid (pJL200) yielded an ROS signal of 154 675 ± 2436 units. In non-inducing media, this value decreased 84% to 25 179 ± ±895 units. Secondly, arg2Δ, ent3Δ, hsp82Δ, idp3Δ and jem1Δ deletion strains expressing WT α-syn each exhibited an ROS signal greater than the control strain. For example, arg2Δ cells expressing WT α-syn exhibited an ROS signal of 203 971 ± 3699 units, which is a 32% increase compared with the control strain (154 675→203 971). The percentage increases in ROS signal compared with the WT control strain were 32% (arg2Δ), 65% (ent3Δ), 21% (hsp82Δ), 62% (idp3Δ) and 37% (jem1Δ). This assay demonstrated that the deletion of genes that protect against WT α-syn toxicity indeed significantly increased the accumulation of ROS. Thirdly, expression of the gene of interest blocked accumulation of ROS, as expected. For example, arg2Δ cells expressing WT α-syn exhibited an ROS signal of 203 971 ± 3699 units, whereas the same cells but with ARG2 added back (pARG2) exhibited a 69% decrease in ROS signal to 62 690 ± ±680 units. On average, adding back the gene of interest in high copy caused a 67% decrease in ROS accumulation in the various deletion strains expressing WT α-syn. The combined results provided further support that these five genes protect cells from WT α-syn toxicity.
Figure 4. ROS accumulation in ent3Δ, idp3Δ, jem1Δ, arg2Δ and hsp82Δ deletion strains expressing WT α-syn. The fluorescence assay using the dye DHR123 was used to measure the ROS accumulation in WT α-synuclein (more ...)
ARG2, ENT3, IDP3, HSP82 and JEM1 fail to inhibit ROS accumulation in cells expressing A30P or A53T
A recent study showed that WT α-syn and A53T, but not A30P, transit through the classical secretory pathway in yeast and target to the plasma membrane (31
), whereas another study showed that the yeast gene YPT1
is a high copy suppressor of the toxicity of WT α-syn and A53T but not of A30P (18
). In light of these findings, we hypothesized that ARG2
would protect cells from WT α-syn and A53T, but not A30P. We thus tested whether these five genes in high copy would protect cells from ROS induced by these two inherited α-syn mutants. The other inherited mutant, E46K, was not evaluated. We have previously shown that A30P and A53T cause ROS accumulation in yeast (15
). Cells were transformed with plasmids for WT α-syn (pTF201), A30P (pTF202) or A53T (pTF203) and with an Open Biosystems plasmid containing one of the five genes of interest, pre-grown in non-inducing media to mid-log phase, shifted into inducing media for 3 h, and assayed for ROS accumulation using DHR123. Matched controls were identically treated cells expressing WT α-syn but without the gene of interest in high copy. The bars in Figure represent the ratio of the two signals: 1 indicates no decrease in ROS, <1 indicates a decrease in ROS and >1 indicates an increase in ROS.
Figure 5. (A) ARG2, ENT3, HSP82, IDP3 and JEM1 in high copy fail to suppress the toxicity of A30P or A53T. The fluorescence assay using DHR123 was used to measure the ROS accumulation in cells expressing various α-synucleins (WT, A30P or A53T). For example, (more ...)
ARG2, ENT3, HSP82, IDP3 and JEM1 in high copy significantly blocked ROS in cells expressing WT α-syn (Fig. A). On average, high copy ARG2, ENT3, HSP82, IDP3 and JEM1 resulted in a 50–60% decrease in ROS accumulation. Strikingly, ARG2, ENT3, HSP82 and IDP3 in high copy had no protective effect on cells expressing A30P or A53T, i.e. for these samples the normalized ROS signal was ≥1. JEM1 in high copy produced a slight decrease in ROS accumulation in cells expressing A30P (S = 0.81 ± 0.09), whereas no appreciable decrease in ROS accumulation was observed for cells expressing A53T. We conclude that although the three α-syns (WT, A30P and A53T) each produce oxidative stress in cells, the pathobiology of these three α-syns must differ (see ‘ENT3 and the vacuole’ in the Discussion section).
To complement the above ROS experiments, western blot analysis was conducted to assess the expression levels of the various α-syns (WT, A30P or A53T) in the FY23 strain overexpressing Arg2p, Ent3p, Hsp82p, Idp3p or Jem1p. A monoclonal antibody that binds to each of the three forms of α-syn was used to visualize α-syn. Pgk1p was used as a loading control. The results are shown in Figure B.
First, consider the western blot of cells overexpressing Arg2p and expressing WT α-syn, A30P or A53T (Fig. B; lanes 1–3). The bands due to WT α-syn (lane 1) and A30P (lane 2) at 14.5 kDa have similar intensities; thus, it may be inferred that the expression levels of these two α-syns are very similar. In contrast, cells overexpressing Arg2p and expressing A53T display an intense band at 54 kDa and a less intense band at 39 kDa, which we assign to aggregated or modified A53T. Secondly, in three of the five experiments with genes in high copy (ARG2
), the A53T protein migrated as three species, i.e. 26, 39 and 54 kDa (lanes 3, 9 and 12). The intensities of these three A53T bands are greater than the intensity of the bands at 14.5 kDa due to monomeric α-syn. The results suggest that under some conditions A53T aggregates accumulate in cells compared with the other two α-syns (WT and A30P). A53T indeed rapidly forms large inclusions when expressed in yeast (15
). Thirdly, in one case, specifically, for cells overexpressing Ent3p, the WT α-syn protein migrated as three high molecular mass species (26, 39 and 54 kDa) (lane 4), whereas A30P and A53T migrated as monomers (lanes 5 and 6). Note that oligomeric forms of WT α-syn have been previously detected using the yeast model (33
). Overall, the western blot results demonstrate that α-syn (WT, A30P and A53T) is indeed expressed in the presence of the various genes in high copy, and that, in general, the A53T variant may accumulate to a greater extent than the other two α-syns. However, the five genes specifically protect only against ROS in cells expressing WT α-syn (Fig. A).
Ent3p alters the localization of GFP-WT α-syn
Given that ENT3
was the strongest suppressor and that it has a human ortholog (epsinR), we sought to determine the effect of Ent3p overexpression on the localization of GFP-WT α-syn by fluorescence microscopy. Yeast cells expressing GFP-WT α-syn displayed bright green fluorescence around the perimeter of the cells at 3 and 12 h of incubation in inducing media (Fig. ; upper panels). This result is consistent with the association of the GFP-WT α-syn protein with the plasma membrane, as has been reported by numerous groups (15
). Identically treated cells expressing GFP-WT α-syn, but also overexpressing Ent3p, displayed a similar membrane association of GFP-WT α-syn at 3 h (Fig. ; lower left panel), whereas cells displayed numerous GFP-WT α-syn inclusions (puncta) at 12 h (Fig. ; lower right panel). For these cells overexpressing Ent3p, it is noteworthy that only 0.5% (n
= 714) of the cells displayed one or more GFP-WT α-syn inclusions at 3 h, whereas 84% (n
= 1562 cells) of the cells displayed one or more GFP-WT α-syn inclusions at 12 h. A possible role of ENT3
in detoxifying yeast cells of WT α-syn is considered in the Discussion section.
Figure 6. ENT3 alters the localization of WT α-syn. The KT2253 strain was transformed with pJL103 (GFP-WT α-syn) and pENT3 or the empty vector pJL200. Cells were pre-grown in non-inducing media until mid-log phase, washed and resuspended in inducing (more ...)