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Effect of endogenous Hsp104 chaperone in yeast models of sporadic and familial Parkinson's disease
The International Journal of Biochemistry & Cell Biology 55 (2014) 87–92
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Effect of endogenous Hsp104 chaperone in yeast models of sporadic and familial Parkinson’s disease Vamshidhar R. Gade 1 , Jay Kardani 1 , Ipsita Roy ∗ Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India
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Article history: Received 30 April 2014 Received in revised form 21 July 2014 Accepted 16 August 2014 Available online 23 August 2014 Keywords: Amyloid fibrils Chaperone Hsp104 Parkinson’s disease Protein aggregation ␣-Synuclein
a b s t r a c t Molecular chaperones constitute a major component of the cellular stress response machinery in neurodegenerative diseases, many of which are characterized by the misfolding and aggregation of endogenous cellular proteins into generic amyloid macrostructures. Heterologous expression of the yeast protein remodelling factor Hsp104 has been proposed as a possible therapeutic approach in such disease conditions. Hsp104 is unique in its ability to act as a protein ‘disaggregase’ by removing smaller units from amyloid fibrils and has no homologue in metazoa. The effect of Hsp104 is strongly modulated by its expression level. We show that at endogenous levels, the presence of Hsp104 has a deleterious effect on protein aggregation in two different strains of yeast. Overexpression of wild-type and mutant human ␣-synuclein in a well-validated yeast model of Parkinson’s disease and in an isogenic Hsp104-deleted strain resulted in lower oxidative stress and reduced damage to cellular proteins in the latter case. This translated to lower cytotoxicity and increased cell viability. Endocytotic defect caused due to aggregation of ␣-syuclein was also rescued in cells lacking Hsp104. Our results show that the effect of overexpression of a chaperone on protein misfolding/aggregation cannot be predicted from its function in the host expression platform. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Aberrant protein misfolding and aggregation are characteristic features of many neurodegenerative disorders, including the movement disorder Parkinson’s disease (PD). PD is characterized by the formation of Lewy bodies in substantia nigra of brain. The signature protein found in these inclusions is ␣-synuclein (Spillantini et al., 1997; Lashuel et al., 2013). PD is a progressively debilitating disorder whose aetiology is mainly unknown. Sporadic cases may result from polymorphisms in SNCA gene (Simón-Sánchez et al., 2009). In cases of familial PD, mutations in ␣-synuclein (A30P, A53T, E46K or H50Q) (Polymeropoulos et al., 1997; Appel-Cresswell et al., 2013) or triplication of wild-type SNCA gene (Singleton et al., 2003) have been correlated with earlier onset of degenerative symptoms. ␣-Synuclein is found localized in the presynaptic terminals. Aggregation of ␣-synuclein is implicated not only in PD but in an assorted family of diseases which are collectively called Lewy body diseases (LBDs) (Galvin et al., 2001).
∗ Corresponding author. Tel.: +91 172 229 206; fax: +91 172 221 4692. E-mail address: [email protected] (I. Roy). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biocel.2014.08.013 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
Heat shock proteins (hsps) form a crucial element of the cellular protein aggregation regulation mechanism (Lashuel et al., 2013). During the initial stages of aggregation, the stress response is activated and the expression of hsps is upregulated. In the latter stages, hsps, along with components of the ubiquitin-proteasome system, are found to be sequestered into these aggregates (Uryu et al., 2006), indicating the function of cellular chaperones in antagonizing the aggregation process. Because of its role as a ‘disaggregase’, the yeast protein remodelling factor Hsp104 has been proposed as a promising candidate to ameliorate aggregation in various protein misfolding disorders (Cashikar et al., 2005; Cushman-Nick et al., 2013). Phenotypic screening identified a small molecule acting on a druggable network and antagonizing ␣-synuclein toxicity in yeast cells (Tardiff et al., 2013). The same candidate molecule was successful in attenuating ␣-synuclein-induced toxicity in mammalian cell and animal models, indicating that the pathobiology of PD is conserved from yeast to higher animals (Tardiff et al., 2013). The yeast model also identified early pathogenic phenotypes in cortical neurons derived from induced pluripotent stem cells (Chung et al., 2013). In this work, we report that the presence of Hsp104 exerts a discriminating effect on the well-validated model of yeast cells expressing wild type or mutant (A53T) ␣-synuclein. As reported earlier (Singh et al., 2013), the deletion of Hsp104 does not result in changes in stress level or viability of yeast cells. The results
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presented here provide evidence that the absence of Hsp104 leads to amelioration of symptoms and improvement in viability of yeast cells expressing ␣-synuclein. 2. Materials and methods 2.1. Materials Saccharomyces cerevisiae BY4742 (MAT␣ his31 leu20 lys20 ura30) (parental and deletion) and W303 (MAT␣ ura3-52 trp12 leu2-3 112 his3-11 ade2-1 can1-100) strains were products of Open Biosystems and were purchased from Saf Labs Pvt. Ltd., India. Nitrocellulose membrane (0.45 m) was purchased from Advanced Microdevices Pvt. Ltd., Ambala, India. Cellulose acetate membrane (0.2 m) was purchased from Sartorius Stedim Biotech GmbH, Goettingen, Germany. Mouse ␣-synuclein antibody, mouse FITC-conjugated antibody and rabbit FITC-conjugated antibody were purchased from Sigma–Aldrich, India. Rabbit anti2,4-dinitrophenol (DNP), Sytox® Orange and FM4-64 dye were purchased from Invitrogen BioServices India Pvt. Ltd., India. All other reagents and chemicals used were of analytical grade or higher.
pre-wetted cellulose acetate or nitrocellulose membrane and probed with ␣-synuclein-specific monoclonal antibody as the primary antibody and mouse FITC-conjugated antibody as the secondary antibody. The membranes were scanned on an image scanner (Typhoon Trio, GE Healthcare, Uppsala, Sweden) operated in the fluorescence mode. The intensity of dots, indicating retention of aggregates on the cellulose acetate membrane or retention of total protein on the nitrocellulose membrane, was quantified using Image QuantTM software (GE Healthcare, Uppsala, Sweden). 2.2.4. Cell toxicity assay Induced cells expressing WT or A53T ␣-synuclein or the empty vector (EV, control) were mixed with Sytox® orange and the fluorescence intensity was monitored as described earlier (Zakrzewska et al., 2007). 2.2.5. Cell viability assay Induced cells (1 × 106 /ml) were serially diluted fivefold and 2 l from each dilution was spotted on SC-URA media containing 2% dextrose agar. Growth was monitored for 2–3 days. Induced cells expressing WT or A53T ␣-synuclein or the empty vector (EV, control) (1 × 103 ) were also streaked on the same media and the colony forming units (CFU) were counted after 2–3 days.
2.2. Methods 2.2.1. Expression of WT-˛-Syn and A53T-˛-Syn in yeast cells Plasmids p426GAL-WT-˛-SYN and p426GAL-A53T-˛-SYN, harbouring gene inserts for wild type (WT-˛-Syn) and mutant (A53T-˛-Syn) human ␣-synuclein, respectively, under the control of Gal1 promoter (Outeiro and Lindquist, 2003), were transformed in S. cerevisiae cells using the lithium acetate-PEG method (Gietz et al., 1992) and grown in SC-URA medium containing 2% (w v−1 ) dextrose at 30 ◦ C till A600 0.6. Protein expression was induced with 2% (w v−1 ) galactose for 8 h. Expression of proteins was confirmed by confocal microscopy (E600 Eclipse, Nikon, Japan), native PAGE and Western blotting. p426GAL-EGFP (empty vector, EV), which lacks the WT- or A53T-˛-SYN sequence, was constructed by the replacement of ˛-SYN region of p426GAL-WT-˛-SYN with a stuffer sequence flanked by restriction sites for HindIII and SpeI. The parent vector (p426GAL-WT-˛-SYN) was digested with HindIII and SpeI to remove WT-˛-SYN. The gel-purified, linearized vector was ligated to the oligonucleotide sequence containing pre-digested HindIII and SpeI restriction sites. The removal of WT-˛-SYN was confirmed by agarose gel electrophoresis. Yeast cells were transformed with the empty vector and treated as above. 2.2.2. Native PAGE and Western blotting Post-induction cell pellets were lysed using acid treated glass beads (Einhauer et al., 2002). Total lysates were centrifuged at 800 × g for 10 min. The amount of protein was estimated by Bradford dye binding method (Bradford, 1976), using bovine serum albumin as the standard protein. The samples were analysed by 12% native-PAGE and scanned using Typhoon Trio (GE Healthcare) in the EGFP mode at ex = 488 nm and em = 526 nm. For expression of RNQ1-EGFP, cells were transformed with pYES2-RNQ1-EGFP as above and induced with 2% (w v−1 ) galactose. Expression of ␣-synuclein was confirmed using ␣-synuclein antibody as the primary antibody and anti-mouse FITC-conjugated antibody as the secondary antibody. For detection of oxidized proteins, the protein bands were visualized using DNP antibody as described earlier (Singh et al., 2013). 2.2.3. Filter retardation assay Lysates of induced cells expressing WT or A53T ␣-synuclein (30 g each of total protein) were vacuum-filtered through a
2.2.6. Endocytosis of FM4-64 Endocytosis of FM4-64 dye by yeast cells was studied over different time periods as described earlier (Outeiro and Lindquist, 2003). 2.2.7. Statistical analysis All values are mean ± standard error of mean (s.e.m.) of at least three independent experiments. Student’s t-test was used to analyse significant difference. A value of p < 0.05 was considered to demonstrate statistical significance. 3. Results and discussion 3.1. Absence of Hsp104 does not result in any visible difference in the expression of wild type or mutant ˛-synuclein Confocal microscopy did not reveal any difference in the expression patterns of WT and A53T ␣-synuclein in S. cerevisiae BY4742 (parental and Hsp104) cells (Fig. 1A) (Outeiro and Lindquist, 2003). In case of parental cells, the protein was initially found in the membrane-bound form and later on, formed cytosolic inclusions. The fraction of protein present in the cytosol was higher than in Hsp104 cells although in this case too, some of the protein was membrane-bound (Fig. 1A). Native PAGE analysis did not reveal any difference in the expression pattern of WT and A53T ␣-synuclein in parental or Hsp104 cells (Fig. 1B). Densitometric analysis showed that the expression level of the mutant (A53T) ␣-synuclein was marginally higher than the wild type protein in Hsp104 cells although not significant (Fig. 1C). Immunoblotting with ␣-synuclein antibody (Fig. 1D) confirmed that the level of expression of both proteins remained unaffected in parental or Hsp104 strains (Fig. 1E). 3.2. Absence of Hsp104 attenuates ˛-synuclein-induced toxicity Since no significant difference in the expression pattern of ␣-synuclein was seen above, the extent of aggregation was determined by filtering the cell lysates through a nitrocellulose membrane (which binds non-specifically to and retains all proteins) and a cellulose acetate membrane (which filters proteins on the basis of size and thus retains high molecular weight aggregates only) and probing the membranes with ␣-synuclein-specific
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Fig. 1. Expression of ␣-synuclein in Saccharomyces cerevisiae BY4742 cells. (A) Expression of protein was monitored in yeast cells by confocal microscopy. Bar = 10 m. (B) Native PAGE analysis of cell lysates. Equal amount of protein was loaded in each lane as compared by estimating protein by Bradford method (Bradford, 1976) and Coomassie staining of the gel after analysis. (C) Densitometric analysis of the band for EGFP signal in each lane of (B) is shown. (D) Immunoblotting of the lysates was performed using ␣-synuclein antibody as the primary antibody. (E) Densitometric analysis of the bands obtained in (D).
Fig. 2. Measurement of toxicity in yeast cells expressing WT-␣-synuclein. (A) Lysates were filtered through a nitrocellulose (total protein) or a cellulose acetate (aggregated protein) membrane to measure the extent of aggregation. Representative blots are shown. **p < 0.01 against parental strain expressing WT-␣-synuclein, N.S., non-significant. (B) Immunoblotting of the lysates was done using DNP (dinitrophenol) antibody as the primary antibody to monitor oxidative damage to proteins. Equal amount of protein was loaded in each lane as compared by estimating protein by Bradford method (Bradford, 1976). (C) Densitometric analysis of the bands obtained in (B). (D) Sytox® assay to determine toxicity in cells of BY4742 strain due to expression of ␣-synuclein. (E) In order to confirm that the cytotoxicity observed was independent of the strain, Sytox® assay was repeated with cells of W303 strain expressing ␣-synuclein. ### p < 0.001 against parental or Hsp104 strain expressing the empty vector, ***p < 0.001 against parental strain expressing the corresponding ␣-synuclein, N.S., non-significant.
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antibody. No significant difference was seen in the total protein expressed in case of parental and Hsp104 cells when comparing the intensities of dots on the nitrocellulose membrane (Fig. 2A). However, the extent of aggregation was significantly lower in case of Hsp104 as compared to parental cells as monitored by the intensities of dots on the cellulose acetate membrane (Fig. 2A), hinting at the role of the chaperone in the aggregation process. Aggregation of ␣-synuclein results in elevated levels of reactive oxygen species (ROS) in yeast and mammalian cells (Xu et al., 2002; Flower et al., 2005). Increased intracellular oxidative stress leads to covalent modification of proteins like carbonylation, either directly or indirectly by their reaction with secondary byproducts of oxidative stress (Singh et al., 2013). Oxidative modification of proteins was detected using anti-DNP antibody (Fig. 2B). Oxidative damage to cellular proteins was higher in cells expressing WT ␣-synuclein as compared to those expressing the mutant (A53T) protein in both parental and Hsp104 cells (Fig. 2C). The damage to proteins was significantly lower in cells deficient in Hsp104. Thus, the presence of the protein remodelling factor Hsp104 exacerbated the oxidative damage to proteins (Fig. 2B) caused due to aggregation of ␣-synuclein (Fig. 2A). Overexpression of ␣-synuclein (WT as well as A53T) in yeast cells has been reported to affect cell growth adversely although no difference has been observed due to mutation (A53T) in the protein (Outeiro and Lindquist, 2003). We measured toxicity in parental and Hsp104 yeast cells expressing the two proteins using the fluorogenic probe Sytox® orange, which penetrates cells whose plasma membrane integrity has been compromised (Zakrzewska et al., 2007). The absence of Hsp104 per se had no effect on the toxicity in yeast cells (comparison of parental and Hsp104 cells containing the empty vector, EV) (Fig. 2D). Expression of ␣-synuclein (WT or A53T) led to significant increase in toxicity in yeast cells although no difference was observed between cells expressing wild type and A53T ␣-synuclein (Fig. 2D). Thus, expression of ␣-synuclein resulted in increased cytotoxicity but mutation in the protein and the resultant aggregation did not lead to any additional toxicity in cells. In cells lacking Hsp104, cytotoxicity was significantly attenuated as compared to parental cells expressing the same protein (either wild type or A53T) (Fig. 2D). Thus, in consonance with the data obtained from reduced aggregation (Fig. 2A) and lower oxidative damage to proteins (Fig. 2B) in cells deficient in Hsp104, toxicity was reduced in these cells. The reduction in toxicity was independent of the strain. This was confirmed by monitoring toxicity in cells of W303 strain. The presence of subdenaturing concentrations of the chaotrope guanidinium hydrochloride does not alter the expression but inhibits the ATP-dependent disaggregase activity of Hsp104 (Grimminger et al., 2004). In the absence of a functional Hsp104, propagation of RNQ1, among other prion proteins, does not occur, and proteins like mutant huntingtin do not aggregate in [rnq− ] cells (Douglas et al., 2009). In case of S. cerevisiae W303 cells treated with 5 mM guanidinium hydrochloride, a similar effect as in BY4742 strain was observed, i.e. lower toxicity was found in these ‘prion-cured’cells which were devoid of Hsp104 activity (Fig. 2E). Also, similar to cells of BY4742 strain, toxicity in cells of W303 strain was found to be independent of the type of protein expressed (i.e. WT- or A53T-␣-synuclein). 3.3. Presence of Hsp104 reduces viability of cells expressing ˛-synuclein PD belongs to the class of misfolding diseases in which cell death occurs largely due to deleterious effects of ␣-synuclein aggregation (Flower et al., 2005). Hence the viability of yeast cells (parental and Hsp104) expressing ␣-synuclein was monitored. No difference in viability was observed when the induced cells expressing WT or A53T protein in either parental or Hsp104 cells were plated on
dextrose (Fig. 3A). However, significant difference was observed in the growth pattern of cells between parental and Hsp104 cells expressing ␣-synuclein (either WT or A53T). The viability of the deletion mutant (Hsp104) was observed to be higher than that of parental cells expressing ␣-synuclein (Fig. 3A). Cytotoxicity was also measured by counting the number of colony forming units (CFU) when a fixed number of induced yeast cells (1 × 103 ) were plated on dextrose. Similar to the data with Sytox orange (Fig. 2D), significant reduction in cell count was observed in case of cells expressing WT or A53T ␣-synuclein as compared with those containing the empty vector (control) (Fig. 3B), confirming cytotoxicity due to expression of ␣-synuclein. No significant difference was seen in the viability of parental and Hsp104 cells expressing either the empty vector or ␣-synuclein. Also, no significant difference in growth was observed between cells expressing either the WT or mutant protein (Fig. 3B). In keeping with the data obtained with cell viability (Fig. 3A), parental cells expressing WT or A53T ␣-synuclein formed less number of CFU than Hsp104 (Fig. 3B). This correlated well with the lower oxidative damage to proteins observed in Hsp104 cells expressing ␣-synuclein (Fig. 2B and C) and reduced uptake of Sytox® orange (Fig. 2D). Thus, it appears that the presence of Hsp104 increases cytotoxicity due to aggregation of ␣-synuclein and its absence exerts beneficial effect on cell growth. This effect of Hsp104 is only marginally dependent on the type of protein expressed, i.e. WT or A53T. We have observed earlier that Hsp104 ablation does not lead to any change in the level of ROS or viability in untransformed yeast cells (Singh et al., 2013). In this work too, we confirm that the absence of Hsp104 per se does not have any deleterious effect on the viability of yeast cells. The differential effect on cell growth observed here is because of the effect of Hsp104 in modulating ␣-synuclein-induced toxicity. No significant difference was found in the number of CFU between ‘cured’ parental (inactive Hsp104) and Hsp104 (Hsp104deleted) cells of BY4742 strain expressing either WT or A53T ␣-synuclein when plated on dextrose (Fig. 3B). In both cases (‘cured’ or Hsp104 cells), the number of colony forming units was significantly higher than in parental cells expressing the corresponding ␣-synuclein. A similar effect was seen in case of W303 cells (Fig. 3C), confirming that the effect observed was independent of the strain. In order to determine if Hsp104 is directly responsible for the observed ameliorative effect in yeast cells expressing ␣-synuclein, the cells were ‘cured’ of Hsp104 activity. The prion status of RNQ1 was monitored by overexpressing the protein in BY4742 strain. In [rnq− ] cells, a significantly higher fraction of the prion protein was seen to exist in the soluble form, as confirmed by confocal microscopy (Fig. 3D) and native PAGE analysis (Fig. 3E). Thus, the disaggregase activity of Hsp104 has an adverse effect on cells expressing ␣-synuclein. It is difficult to pinpoint the actual role of prions in the aggregation process as the absence of Hsp104 is known to express proteins like RNQ1 in the non-prion, i.e. soluble, form (Meriin et al., 2002). Hsp104 resolves larger aggregates into oligomers. These oligomers formed upon cleavage of aggregates may act as ‘seeds’ or nuclei and are likely to be more toxic to the cells than the mature aggregates formed in the absence of Hsp104. 3.4. Absence of Hsp104 rescues endocytotic defect in yeast cells expressing ˛-synuclein Misfolding and aggregation of intracellular proteins result in the disruption of many cellular functions, including protein trafficking and endocytosis. Internalization of the lipophilic styryl dye FM4-64 has been used to monitor vesicular trafficking and endocytosis in yeast cell models of Huntington’s disease (Meriin et al., 2003) and Parkinson’s disease (Outeiro and Lindquist, 2003). FM464 initially stains the plasma membrane followed by cytoplasmic
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Fig. 3. Effect of expression of ␣-synuclein on cell survival. (A) Cell viability assay. Arrow indicates direction of dilution. (B) Comparison of colony forming units in parental (empty bars), Hsp104 (filled bars) and prion-‘cured’ parental (grey bars) cells of BY4742 strain expressing empty vector or ␣-synuclein. ### p < 0.001 against parental or Hsp104 strain expressing the empty vector, ***p < 0.001 against corresponding parental strain expressing corresponding ␣-synuclein. (C) Comparison of colony forming units in parental (empty bars) and prion-‘cured’ parental (grey bars) cells of W303 strain expressing ␣-synuclein. ***p < 0.001 against corresponding parental strain expressing corresponding ␣-synuclein. Expression of RNQ1-EGFP in Saccharomyces cerevisiae BY4742 cells was monitored by (D) confocal microscopy and (E) native PAGE analysis. Bar = 2 m.
compartments and finally vacuolar membrane by an endocytotic mechanism. Uptake of the dye was monitored in parental and Hsp104 cells expressing WT and A53T ␣-synuclein after different periods of incubation. At 15 min, a marginally higher fraction of cells expressing WT protein internalized the dye as compared to
those expressing the mutant protein (Fig. 4A), signifying delayed internalization in the latter case. Hsp104 cells expressing WT ␣synuclein, on the other hand, displayed delayed internalization of FM4-64 as compared to cells of the same strain expressing A53T ␣-synuclein although the difference was not significant (Fig. 4A).
Fig. 4. (A) Representative images of internalization of FM4-64 in cells expressing ␣-synuclein after 15 min of incubation with the dye. (B) The number of cells internalizing FM4-64 was calculated as a fraction of total number of cells expressing EGFP-␣-synuclein. The value of 100% assumes that all cells expressing EGFP-␣-synuclein have internalized FM4-64. A total of twenty windows (∼40 cells each) were counted each for parental (empty bars) and Hsp104 (filled bars) cells. *p < 0.05 against parental strain expressing corresponding ␣-synuclein. Bar = 100 m.
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Comparison of cells expressing WT ␣-synuclein indicated that the parental cells exhibited a significant lower level of internalization of FM4-64 dye after 15 min as compared to Hsp104 cells (Fig. 4B). A similar result was seen in case of cells expressing A53T ␣-synuclein (Fig. 4B). Since faster internalization of the lipophilic dye indicates reduction in the fraction of aggregates, this correlates well with lower oxidative damage, reduced cytotoxicity and increased cell viability observed in case of Hsp104 cells expressing either WT or A53T ␣-synuclein. How do our results correlate with the proposed strategy of employing Hsp104 as a therapeutic chaperone in protein misfolding diseases? Almost all the available reports evaluate the role of overexpressed Hsp104 in cells. This could and does activate compensatory mechanisms and does not lead to unambiguous elucidation of the function which the chaperone might play. For example, a recent report of overexpression of wild type Hsp104 in yeast cells has shown marginally higher toxicity due to the heterologous expression of ␣-synuclein (Jackrel et al., 2014). Overexpression of Hsp104 in the brain of a rat model of PD rescued loss of tyrosine hydroxylase-positive cells and reduced the formation of phosphorylated inclusions (Lo Bianco et al., 2008). Overexpression of the chaperone also reduced the formation of ␣-synuclein fibrils in a bacterial system (Kong et al., 2005). However, in yeast cells engineered to synthesize the compatible solute mannosylglycerate, stabilization of heterologous ␣-synuclein was seen without upregulation of Hsp104 (Faria et al., 2013). Overexpression of Hsp104 enhanced external eye degeneration in fruit flies expressing the full-length Machado-Joseph-Disease protein containing expanded polyglutamine tract (78Q) (Cushman-Nick et al., 2013). Different amounts of detergent-soluble and -insoluble fractions were formed, depending on the presence/absence of Hsp104. The presence of an inactive variant (Hsp104DPLDWB ) rescued retinal degeneration (Cushman-Nick et al., 2013), confirming the role of ATPase function in the activity of Hsp104 as a modulator of degeneration. Similar to our case, the level of expression of the protein did not change but the presence or absence of Hsp104 had a differential effect on cellular oxidative stress, damage to the cellular proteome and cell viability. The difference between the molecular function of a gene and its emergent property, when expressed in different organisms, does not prevent the use of such organisms as models for mimicking disease conditions (McGary et al., 2010). Indeed, the appreciation of such differences can lead to the logical development of orthologous phenotypes (phenologs), which allows their use as genetic screens, for example (Chung et al., 2013; Tardiff et al., 2013), to identify novel drug targets and/or candidates for many recalcitrant human diseases. Acknowledgements The authors are thankful to Prof. Susan Lindquist, Massachusetts Institute of Technology, Massachusetts, USA, for the plasmid constructs p426GAL-WT␣-SYN and p426GAL-A53T˛-SYN, to Prof. Douglas Cyr, University of North Carolina, USA, for pRS315-RNQ1mRFP and to Prof. Michael Sherman, Boston University School of Medicine, Boston, USA, for pYES2-FLAG-25Q-EGFP. Further manipulations to generate pYES2-RNQ1-EGFP were carried out by Vishal Patel. References Appel-Cresswell S, Vilarino-Guell C, Encarnacion M, Sherman H, Yu I, Shah B, et al. Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson’s disease. Mov Disord 2013;28:811–3.
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Effect of endogenous Hsp104 chaperone in yeast models of sporadic and familial Parkinson’s disease Vamshidhar R. Gade 1 , Jay Kardani 1 , Ipsita Roy ∗ Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India
a r t i c l e
i n f o
Article history: Received 30 April 2014 Received in revised form 21 July 2014 Accepted 16 August 2014 Available online 23 August 2014 Keywords: Amyloid fibrils Chaperone Hsp104 Parkinson’s disease Protein aggregation ␣-Synuclein
a b s t r a c t Molecular chaperones constitute a major component of the cellular stress response machinery in neurodegenerative diseases, many of which are characterized by the misfolding and aggregation of endogenous cellular proteins into generic amyloid macrostructures. Heterologous expression of the yeast protein remodelling factor Hsp104 has been proposed as a possible therapeutic approach in such disease conditions. Hsp104 is unique in its ability to act as a protein ‘disaggregase’ by removing smaller units from amyloid fibrils and has no homologue in metazoa. The effect of Hsp104 is strongly modulated by its expression level. We show that at endogenous levels, the presence of Hsp104 has a deleterious effect on protein aggregation in two different strains of yeast. Overexpression of wild-type and mutant human ␣-synuclein in a well-validated yeast model of Parkinson’s disease and in an isogenic Hsp104-deleted strain resulted in lower oxidative stress and reduced damage to cellular proteins in the latter case. This translated to lower cytotoxicity and increased cell viability. Endocytotic defect caused due to aggregation of ␣-syuclein was also rescued in cells lacking Hsp104. Our results show that the effect of overexpression of a chaperone on protein misfolding/aggregation cannot be predicted from its function in the host expression platform. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Aberrant protein misfolding and aggregation are characteristic features of many neurodegenerative disorders, including the movement disorder Parkinson’s disease (PD). PD is characterized by the formation of Lewy bodies in substantia nigra of brain. The signature protein found in these inclusions is ␣-synuclein (Spillantini et al., 1997; Lashuel et al., 2013). PD is a progressively debilitating disorder whose aetiology is mainly unknown. Sporadic cases may result from polymorphisms in SNCA gene (Simón-Sánchez et al., 2009). In cases of familial PD, mutations in ␣-synuclein (A30P, A53T, E46K or H50Q) (Polymeropoulos et al., 1997; Appel-Cresswell et al., 2013) or triplication of wild-type SNCA gene (Singleton et al., 2003) have been correlated with earlier onset of degenerative symptoms. ␣-Synuclein is found localized in the presynaptic terminals. Aggregation of ␣-synuclein is implicated not only in PD but in an assorted family of diseases which are collectively called Lewy body diseases (LBDs) (Galvin et al., 2001).
∗ Corresponding author. Tel.: +91 172 229 206; fax: +91 172 221 4692. E-mail address: [email protected] (I. Roy). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biocel.2014.08.013 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
Heat shock proteins (hsps) form a crucial element of the cellular protein aggregation regulation mechanism (Lashuel et al., 2013). During the initial stages of aggregation, the stress response is activated and the expression of hsps is upregulated. In the latter stages, hsps, along with components of the ubiquitin-proteasome system, are found to be sequestered into these aggregates (Uryu et al., 2006), indicating the function of cellular chaperones in antagonizing the aggregation process. Because of its role as a ‘disaggregase’, the yeast protein remodelling factor Hsp104 has been proposed as a promising candidate to ameliorate aggregation in various protein misfolding disorders (Cashikar et al., 2005; Cushman-Nick et al., 2013). Phenotypic screening identified a small molecule acting on a druggable network and antagonizing ␣-synuclein toxicity in yeast cells (Tardiff et al., 2013). The same candidate molecule was successful in attenuating ␣-synuclein-induced toxicity in mammalian cell and animal models, indicating that the pathobiology of PD is conserved from yeast to higher animals (Tardiff et al., 2013). The yeast model also identified early pathogenic phenotypes in cortical neurons derived from induced pluripotent stem cells (Chung et al., 2013). In this work, we report that the presence of Hsp104 exerts a discriminating effect on the well-validated model of yeast cells expressing wild type or mutant (A53T) ␣-synuclein. As reported earlier (Singh et al., 2013), the deletion of Hsp104 does not result in changes in stress level or viability of yeast cells. The results
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presented here provide evidence that the absence of Hsp104 leads to amelioration of symptoms and improvement in viability of yeast cells expressing ␣-synuclein. 2. Materials and methods 2.1. Materials Saccharomyces cerevisiae BY4742 (MAT␣ his31 leu20 lys20 ura30) (parental and deletion) and W303 (MAT␣ ura3-52 trp12 leu2-3 112 his3-11 ade2-1 can1-100) strains were products of Open Biosystems and were purchased from Saf Labs Pvt. Ltd., India. Nitrocellulose membrane (0.45 m) was purchased from Advanced Microdevices Pvt. Ltd., Ambala, India. Cellulose acetate membrane (0.2 m) was purchased from Sartorius Stedim Biotech GmbH, Goettingen, Germany. Mouse ␣-synuclein antibody, mouse FITC-conjugated antibody and rabbit FITC-conjugated antibody were purchased from Sigma–Aldrich, India. Rabbit anti2,4-dinitrophenol (DNP), Sytox® Orange and FM4-64 dye were purchased from Invitrogen BioServices India Pvt. Ltd., India. All other reagents and chemicals used were of analytical grade or higher.
pre-wetted cellulose acetate or nitrocellulose membrane and probed with ␣-synuclein-specific monoclonal antibody as the primary antibody and mouse FITC-conjugated antibody as the secondary antibody. The membranes were scanned on an image scanner (Typhoon Trio, GE Healthcare, Uppsala, Sweden) operated in the fluorescence mode. The intensity of dots, indicating retention of aggregates on the cellulose acetate membrane or retention of total protein on the nitrocellulose membrane, was quantified using Image QuantTM software (GE Healthcare, Uppsala, Sweden). 2.2.4. Cell toxicity assay Induced cells expressing WT or A53T ␣-synuclein or the empty vector (EV, control) were mixed with Sytox® orange and the fluorescence intensity was monitored as described earlier (Zakrzewska et al., 2007). 2.2.5. Cell viability assay Induced cells (1 × 106 /ml) were serially diluted fivefold and 2 l from each dilution was spotted on SC-URA media containing 2% dextrose agar. Growth was monitored for 2–3 days. Induced cells expressing WT or A53T ␣-synuclein or the empty vector (EV, control) (1 × 103 ) were also streaked on the same media and the colony forming units (CFU) were counted after 2–3 days.
2.2. Methods 2.2.1. Expression of WT-˛-Syn and A53T-˛-Syn in yeast cells Plasmids p426GAL-WT-˛-SYN and p426GAL-A53T-˛-SYN, harbouring gene inserts for wild type (WT-˛-Syn) and mutant (A53T-˛-Syn) human ␣-synuclein, respectively, under the control of Gal1 promoter (Outeiro and Lindquist, 2003), were transformed in S. cerevisiae cells using the lithium acetate-PEG method (Gietz et al., 1992) and grown in SC-URA medium containing 2% (w v−1 ) dextrose at 30 ◦ C till A600 0.6. Protein expression was induced with 2% (w v−1 ) galactose for 8 h. Expression of proteins was confirmed by confocal microscopy (E600 Eclipse, Nikon, Japan), native PAGE and Western blotting. p426GAL-EGFP (empty vector, EV), which lacks the WT- or A53T-˛-SYN sequence, was constructed by the replacement of ˛-SYN region of p426GAL-WT-˛-SYN with a stuffer sequence flanked by restriction sites for HindIII and SpeI. The parent vector (p426GAL-WT-˛-SYN) was digested with HindIII and SpeI to remove WT-˛-SYN. The gel-purified, linearized vector was ligated to the oligonucleotide sequence containing pre-digested HindIII and SpeI restriction sites. The removal of WT-˛-SYN was confirmed by agarose gel electrophoresis. Yeast cells were transformed with the empty vector and treated as above. 2.2.2. Native PAGE and Western blotting Post-induction cell pellets were lysed using acid treated glass beads (Einhauer et al., 2002). Total lysates were centrifuged at 800 × g for 10 min. The amount of protein was estimated by Bradford dye binding method (Bradford, 1976), using bovine serum albumin as the standard protein. The samples were analysed by 12% native-PAGE and scanned using Typhoon Trio (GE Healthcare) in the EGFP mode at ex = 488 nm and em = 526 nm. For expression of RNQ1-EGFP, cells were transformed with pYES2-RNQ1-EGFP as above and induced with 2% (w v−1 ) galactose. Expression of ␣-synuclein was confirmed using ␣-synuclein antibody as the primary antibody and anti-mouse FITC-conjugated antibody as the secondary antibody. For detection of oxidized proteins, the protein bands were visualized using DNP antibody as described earlier (Singh et al., 2013). 2.2.3. Filter retardation assay Lysates of induced cells expressing WT or A53T ␣-synuclein (30 g each of total protein) were vacuum-filtered through a
2.2.6. Endocytosis of FM4-64 Endocytosis of FM4-64 dye by yeast cells was studied over different time periods as described earlier (Outeiro and Lindquist, 2003). 2.2.7. Statistical analysis All values are mean ± standard error of mean (s.e.m.) of at least three independent experiments. Student’s t-test was used to analyse significant difference. A value of p < 0.05 was considered to demonstrate statistical significance. 3. Results and discussion 3.1. Absence of Hsp104 does not result in any visible difference in the expression of wild type or mutant ˛-synuclein Confocal microscopy did not reveal any difference in the expression patterns of WT and A53T ␣-synuclein in S. cerevisiae BY4742 (parental and Hsp104) cells (Fig. 1A) (Outeiro and Lindquist, 2003). In case of parental cells, the protein was initially found in the membrane-bound form and later on, formed cytosolic inclusions. The fraction of protein present in the cytosol was higher than in Hsp104 cells although in this case too, some of the protein was membrane-bound (Fig. 1A). Native PAGE analysis did not reveal any difference in the expression pattern of WT and A53T ␣-synuclein in parental or Hsp104 cells (Fig. 1B). Densitometric analysis showed that the expression level of the mutant (A53T) ␣-synuclein was marginally higher than the wild type protein in Hsp104 cells although not significant (Fig. 1C). Immunoblotting with ␣-synuclein antibody (Fig. 1D) confirmed that the level of expression of both proteins remained unaffected in parental or Hsp104 strains (Fig. 1E). 3.2. Absence of Hsp104 attenuates ˛-synuclein-induced toxicity Since no significant difference in the expression pattern of ␣-synuclein was seen above, the extent of aggregation was determined by filtering the cell lysates through a nitrocellulose membrane (which binds non-specifically to and retains all proteins) and a cellulose acetate membrane (which filters proteins on the basis of size and thus retains high molecular weight aggregates only) and probing the membranes with ␣-synuclein-specific
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Fig. 1. Expression of ␣-synuclein in Saccharomyces cerevisiae BY4742 cells. (A) Expression of protein was monitored in yeast cells by confocal microscopy. Bar = 10 m. (B) Native PAGE analysis of cell lysates. Equal amount of protein was loaded in each lane as compared by estimating protein by Bradford method (Bradford, 1976) and Coomassie staining of the gel after analysis. (C) Densitometric analysis of the band for EGFP signal in each lane of (B) is shown. (D) Immunoblotting of the lysates was performed using ␣-synuclein antibody as the primary antibody. (E) Densitometric analysis of the bands obtained in (D).
Fig. 2. Measurement of toxicity in yeast cells expressing WT-␣-synuclein. (A) Lysates were filtered through a nitrocellulose (total protein) or a cellulose acetate (aggregated protein) membrane to measure the extent of aggregation. Representative blots are shown. **p < 0.01 against parental strain expressing WT-␣-synuclein, N.S., non-significant. (B) Immunoblotting of the lysates was done using DNP (dinitrophenol) antibody as the primary antibody to monitor oxidative damage to proteins. Equal amount of protein was loaded in each lane as compared by estimating protein by Bradford method (Bradford, 1976). (C) Densitometric analysis of the bands obtained in (B). (D) Sytox® assay to determine toxicity in cells of BY4742 strain due to expression of ␣-synuclein. (E) In order to confirm that the cytotoxicity observed was independent of the strain, Sytox® assay was repeated with cells of W303 strain expressing ␣-synuclein. ### p < 0.001 against parental or Hsp104 strain expressing the empty vector, ***p < 0.001 against parental strain expressing the corresponding ␣-synuclein, N.S., non-significant.
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antibody. No significant difference was seen in the total protein expressed in case of parental and Hsp104 cells when comparing the intensities of dots on the nitrocellulose membrane (Fig. 2A). However, the extent of aggregation was significantly lower in case of Hsp104 as compared to parental cells as monitored by the intensities of dots on the cellulose acetate membrane (Fig. 2A), hinting at the role of the chaperone in the aggregation process. Aggregation of ␣-synuclein results in elevated levels of reactive oxygen species (ROS) in yeast and mammalian cells (Xu et al., 2002; Flower et al., 2005). Increased intracellular oxidative stress leads to covalent modification of proteins like carbonylation, either directly or indirectly by their reaction with secondary byproducts of oxidative stress (Singh et al., 2013). Oxidative modification of proteins was detected using anti-DNP antibody (Fig. 2B). Oxidative damage to cellular proteins was higher in cells expressing WT ␣-synuclein as compared to those expressing the mutant (A53T) protein in both parental and Hsp104 cells (Fig. 2C). The damage to proteins was significantly lower in cells deficient in Hsp104. Thus, the presence of the protein remodelling factor Hsp104 exacerbated the oxidative damage to proteins (Fig. 2B) caused due to aggregation of ␣-synuclein (Fig. 2A). Overexpression of ␣-synuclein (WT as well as A53T) in yeast cells has been reported to affect cell growth adversely although no difference has been observed due to mutation (A53T) in the protein (Outeiro and Lindquist, 2003). We measured toxicity in parental and Hsp104 yeast cells expressing the two proteins using the fluorogenic probe Sytox® orange, which penetrates cells whose plasma membrane integrity has been compromised (Zakrzewska et al., 2007). The absence of Hsp104 per se had no effect on the toxicity in yeast cells (comparison of parental and Hsp104 cells containing the empty vector, EV) (Fig. 2D). Expression of ␣-synuclein (WT or A53T) led to significant increase in toxicity in yeast cells although no difference was observed between cells expressing wild type and A53T ␣-synuclein (Fig. 2D). Thus, expression of ␣-synuclein resulted in increased cytotoxicity but mutation in the protein and the resultant aggregation did not lead to any additional toxicity in cells. In cells lacking Hsp104, cytotoxicity was significantly attenuated as compared to parental cells expressing the same protein (either wild type or A53T) (Fig. 2D). Thus, in consonance with the data obtained from reduced aggregation (Fig. 2A) and lower oxidative damage to proteins (Fig. 2B) in cells deficient in Hsp104, toxicity was reduced in these cells. The reduction in toxicity was independent of the strain. This was confirmed by monitoring toxicity in cells of W303 strain. The presence of subdenaturing concentrations of the chaotrope guanidinium hydrochloride does not alter the expression but inhibits the ATP-dependent disaggregase activity of Hsp104 (Grimminger et al., 2004). In the absence of a functional Hsp104, propagation of RNQ1, among other prion proteins, does not occur, and proteins like mutant huntingtin do not aggregate in [rnq− ] cells (Douglas et al., 2009). In case of S. cerevisiae W303 cells treated with 5 mM guanidinium hydrochloride, a similar effect as in BY4742 strain was observed, i.e. lower toxicity was found in these ‘prion-cured’cells which were devoid of Hsp104 activity (Fig. 2E). Also, similar to cells of BY4742 strain, toxicity in cells of W303 strain was found to be independent of the type of protein expressed (i.e. WT- or A53T-␣-synuclein). 3.3. Presence of Hsp104 reduces viability of cells expressing ˛-synuclein PD belongs to the class of misfolding diseases in which cell death occurs largely due to deleterious effects of ␣-synuclein aggregation (Flower et al., 2005). Hence the viability of yeast cells (parental and Hsp104) expressing ␣-synuclein was monitored. No difference in viability was observed when the induced cells expressing WT or A53T protein in either parental or Hsp104 cells were plated on
dextrose (Fig. 3A). However, significant difference was observed in the growth pattern of cells between parental and Hsp104 cells expressing ␣-synuclein (either WT or A53T). The viability of the deletion mutant (Hsp104) was observed to be higher than that of parental cells expressing ␣-synuclein (Fig. 3A). Cytotoxicity was also measured by counting the number of colony forming units (CFU) when a fixed number of induced yeast cells (1 × 103 ) were plated on dextrose. Similar to the data with Sytox orange (Fig. 2D), significant reduction in cell count was observed in case of cells expressing WT or A53T ␣-synuclein as compared with those containing the empty vector (control) (Fig. 3B), confirming cytotoxicity due to expression of ␣-synuclein. No significant difference was seen in the viability of parental and Hsp104 cells expressing either the empty vector or ␣-synuclein. Also, no significant difference in growth was observed between cells expressing either the WT or mutant protein (Fig. 3B). In keeping with the data obtained with cell viability (Fig. 3A), parental cells expressing WT or A53T ␣-synuclein formed less number of CFU than Hsp104 (Fig. 3B). This correlated well with the lower oxidative damage to proteins observed in Hsp104 cells expressing ␣-synuclein (Fig. 2B and C) and reduced uptake of Sytox® orange (Fig. 2D). Thus, it appears that the presence of Hsp104 increases cytotoxicity due to aggregation of ␣-synuclein and its absence exerts beneficial effect on cell growth. This effect of Hsp104 is only marginally dependent on the type of protein expressed, i.e. WT or A53T. We have observed earlier that Hsp104 ablation does not lead to any change in the level of ROS or viability in untransformed yeast cells (Singh et al., 2013). In this work too, we confirm that the absence of Hsp104 per se does not have any deleterious effect on the viability of yeast cells. The differential effect on cell growth observed here is because of the effect of Hsp104 in modulating ␣-synuclein-induced toxicity. No significant difference was found in the number of CFU between ‘cured’ parental (inactive Hsp104) and Hsp104 (Hsp104deleted) cells of BY4742 strain expressing either WT or A53T ␣-synuclein when plated on dextrose (Fig. 3B). In both cases (‘cured’ or Hsp104 cells), the number of colony forming units was significantly higher than in parental cells expressing the corresponding ␣-synuclein. A similar effect was seen in case of W303 cells (Fig. 3C), confirming that the effect observed was independent of the strain. In order to determine if Hsp104 is directly responsible for the observed ameliorative effect in yeast cells expressing ␣-synuclein, the cells were ‘cured’ of Hsp104 activity. The prion status of RNQ1 was monitored by overexpressing the protein in BY4742 strain. In [rnq− ] cells, a significantly higher fraction of the prion protein was seen to exist in the soluble form, as confirmed by confocal microscopy (Fig. 3D) and native PAGE analysis (Fig. 3E). Thus, the disaggregase activity of Hsp104 has an adverse effect on cells expressing ␣-synuclein. It is difficult to pinpoint the actual role of prions in the aggregation process as the absence of Hsp104 is known to express proteins like RNQ1 in the non-prion, i.e. soluble, form (Meriin et al., 2002). Hsp104 resolves larger aggregates into oligomers. These oligomers formed upon cleavage of aggregates may act as ‘seeds’ or nuclei and are likely to be more toxic to the cells than the mature aggregates formed in the absence of Hsp104. 3.4. Absence of Hsp104 rescues endocytotic defect in yeast cells expressing ˛-synuclein Misfolding and aggregation of intracellular proteins result in the disruption of many cellular functions, including protein trafficking and endocytosis. Internalization of the lipophilic styryl dye FM4-64 has been used to monitor vesicular trafficking and endocytosis in yeast cell models of Huntington’s disease (Meriin et al., 2003) and Parkinson’s disease (Outeiro and Lindquist, 2003). FM464 initially stains the plasma membrane followed by cytoplasmic
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Fig. 3. Effect of expression of ␣-synuclein on cell survival. (A) Cell viability assay. Arrow indicates direction of dilution. (B) Comparison of colony forming units in parental (empty bars), Hsp104 (filled bars) and prion-‘cured’ parental (grey bars) cells of BY4742 strain expressing empty vector or ␣-synuclein. ### p < 0.001 against parental or Hsp104 strain expressing the empty vector, ***p < 0.001 against corresponding parental strain expressing corresponding ␣-synuclein. (C) Comparison of colony forming units in parental (empty bars) and prion-‘cured’ parental (grey bars) cells of W303 strain expressing ␣-synuclein. ***p < 0.001 against corresponding parental strain expressing corresponding ␣-synuclein. Expression of RNQ1-EGFP in Saccharomyces cerevisiae BY4742 cells was monitored by (D) confocal microscopy and (E) native PAGE analysis. Bar = 2 m.
compartments and finally vacuolar membrane by an endocytotic mechanism. Uptake of the dye was monitored in parental and Hsp104 cells expressing WT and A53T ␣-synuclein after different periods of incubation. At 15 min, a marginally higher fraction of cells expressing WT protein internalized the dye as compared to
those expressing the mutant protein (Fig. 4A), signifying delayed internalization in the latter case. Hsp104 cells expressing WT ␣synuclein, on the other hand, displayed delayed internalization of FM4-64 as compared to cells of the same strain expressing A53T ␣-synuclein although the difference was not significant (Fig. 4A).
Fig. 4. (A) Representative images of internalization of FM4-64 in cells expressing ␣-synuclein after 15 min of incubation with the dye. (B) The number of cells internalizing FM4-64 was calculated as a fraction of total number of cells expressing EGFP-␣-synuclein. The value of 100% assumes that all cells expressing EGFP-␣-synuclein have internalized FM4-64. A total of twenty windows (∼40 cells each) were counted each for parental (empty bars) and Hsp104 (filled bars) cells. *p < 0.05 against parental strain expressing corresponding ␣-synuclein. Bar = 100 m.
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Comparison of cells expressing WT ␣-synuclein indicated that the parental cells exhibited a significant lower level of internalization of FM4-64 dye after 15 min as compared to Hsp104 cells (Fig. 4B). A similar result was seen in case of cells expressing A53T ␣-synuclein (Fig. 4B). Since faster internalization of the lipophilic dye indicates reduction in the fraction of aggregates, this correlates well with lower oxidative damage, reduced cytotoxicity and increased cell viability observed in case of Hsp104 cells expressing either WT or A53T ␣-synuclein. How do our results correlate with the proposed strategy of employing Hsp104 as a therapeutic chaperone in protein misfolding diseases? Almost all the available reports evaluate the role of overexpressed Hsp104 in cells. This could and does activate compensatory mechanisms and does not lead to unambiguous elucidation of the function which the chaperone might play. For example, a recent report of overexpression of wild type Hsp104 in yeast cells has shown marginally higher toxicity due to the heterologous expression of ␣-synuclein (Jackrel et al., 2014). Overexpression of Hsp104 in the brain of a rat model of PD rescued loss of tyrosine hydroxylase-positive cells and reduced the formation of phosphorylated inclusions (Lo Bianco et al., 2008). Overexpression of the chaperone also reduced the formation of ␣-synuclein fibrils in a bacterial system (Kong et al., 2005). However, in yeast cells engineered to synthesize the compatible solute mannosylglycerate, stabilization of heterologous ␣-synuclein was seen without upregulation of Hsp104 (Faria et al., 2013). Overexpression of Hsp104 enhanced external eye degeneration in fruit flies expressing the full-length Machado-Joseph-Disease protein containing expanded polyglutamine tract (78Q) (Cushman-Nick et al., 2013). Different amounts of detergent-soluble and -insoluble fractions were formed, depending on the presence/absence of Hsp104. The presence of an inactive variant (Hsp104DPLDWB ) rescued retinal degeneration (Cushman-Nick et al., 2013), confirming the role of ATPase function in the activity of Hsp104 as a modulator of degeneration. Similar to our case, the level of expression of the protein did not change but the presence or absence of Hsp104 had a differential effect on cellular oxidative stress, damage to the cellular proteome and cell viability. The difference between the molecular function of a gene and its emergent property, when expressed in different organisms, does not prevent the use of such organisms as models for mimicking disease conditions (McGary et al., 2010). Indeed, the appreciation of such differences can lead to the logical development of orthologous phenotypes (phenologs), which allows their use as genetic screens, for example (Chung et al., 2013; Tardiff et al., 2013), to identify novel drug targets and/or candidates for many recalcitrant human diseases. Acknowledgements The authors are thankful to Prof. Susan Lindquist, Massachusetts Institute of Technology, Massachusetts, USA, for the plasmid constructs p426GAL-WT␣-SYN and p426GAL-A53T˛-SYN, to Prof. Douglas Cyr, University of North Carolina, USA, for pRS315-RNQ1mRFP and to Prof. Michael Sherman, Boston University School of Medicine, Boston, USA, for pYES2-FLAG-25Q-EGFP. Further manipulations to generate pYES2-RNQ1-EGFP were carried out by Vishal Patel. References Appel-Cresswell S, Vilarino-Guell C, Encarnacion M, Sherman H, Yu I, Shah B, et al. Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson’s disease. Mov Disord 2013;28:811–3.
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