Having established the validity of our method to uncover connections between otherwise disparate high-throughput datasets, we applied ResponseNet to investigate the cellular toxicity associated with alpha-synuclein (α-syn). α-syn is a small lipid-binding protein that is natively unfolded when not bound to lipids and prone to forming toxic oligomers 23
. It that has been implicated in several neurodegenerative disorders, most particularly Parkinson disease (PD). α-syn is the main component of Lewy bodies, cytoplasmic proteinaceous inclusions that are a hallmark of PD 24
; locus duplication or triplication of α-syn lead to familial forms of PD 25,26
, and increased expression of α-syn leads to neurodegeneration in several animal models 27
. α-syn is linked to alterations in vesicle trafficking 12,28
and mitochondrial function 29
, yet despite immense efforts, the cellular pathways by which α-syn leads to cell death are just beginning to be uncovered.
The yeast S. cerevisiae
provides a powerful system for studying the molecular basis of α-syn toxicity that result from its intrinsic physical properties. Expression of human α-syn in yeast yields several dosage-dependent defects that are also found in mammalian systems, such as lipid droplet accumulation in the cytosol, the production of reactive oxygen species and impairment of the ubiquitin-proteasome system 11
. An initial overexpression screen in yeast for genes that modify α-syn toxicity tested 2,000 strains and identified a class of genes functioning in ER to Golgi vesicle trafficking, leading to the observation that α-syn causes an ER to Golgi vesicle trafficking block. One of these genes, Ypt1/Rab1, a GTPase protein, was tested in neuronal models of PD and was found to rescue dopaminergic neurons from α-syn toxicity 12
We now report other results from that screen and the results of screening an additional set of 3,500 overexpression yeast strains, thereby covering in total 85% of the yeast proteome. We identified a diverse group of genes including 54 suppressors and 23 enhancers of α-syn toxicity, many with clear human orthologs (). Major classes of genes that emerged include vesicle-trafficking genes, kinases and phosphatases, ubiquitin-related proteins, transcriptional regulators, manganese transporters, and trehalose biosynthesis genes. Significantly enriched GO categories included ER to Golgi vesicle-mediated transport (12%, p=6.2*10−5
), phosphatases (9.1%, p=0.0028) and transcription factors (6.5%, p=0.047). While the identification of additional vesicle trafficking and ubiquitin-related genes is consistent with the defects caused by α-syn expression in yeast, the identification of trehalose biosynthesis genes and manganese transporters was new and intriguing. Trehalose was recently shown to promote the clearance of misfolded mutant α-syn 30
, and manganese exposure has been linked with Parkinson-like symptoms albeit with a distinct underlying pathology31
. Notably, another suppressor we identified is homologous to the human PD gene PARK9.
Yeast genes that modify α-syn toxicity when overexpressed.
Park9 and the human homologs of seven other genetic modifiers from diverse functional classes (Hrd1, Ubp3, Pde2, Cdc5, Yck3, Sit4 and Pmr1) were found to be efficacious in neuronal models, validating the yeast model as meaningful to α-syn toxicity in neurons (Gitler et al.; manuscript submitted). The genes identified by the screen therefore begin to unravel the surprisingly multifaceted toxicity of α-syn. Importantly, they provide novel causal relations between α-syn expression and toxicities previously associated with PD but not specifically linked to α-syn. A detailed description of the various gene classes and their potential relation to PD appears in the Supplementary Text.
The transcriptional profile occurring in response to α-syn toxicity was determined in a separate study (Supplementary Text; Su et al.; manuscript submitted). Up-regulated genes prominently included genes with oxidoreductase activities (13%, p<10−9
). Down-regulated genes included ribosomal genes (28%, p<10−30
), as commonly observed under stress 32
. More specific to α-syn toxicity, the down-regulated genes were strikingly enriched for genes encoding proteins localized to the mitochondria (60%, p<10−44
) and for genes involved in generation of precursor metabolites and energy (18%, p<10−15
The genetic and transcriptional data obtained in this model system exemplify both the power and the limitations of the current approaches. These technologies reveal the wide range of cellular functions that are altered by α-syn expression. Yet the precise roles of the genetic hits and differentially expressed genes in the cellular response are unclear. For example, we checked whether the ubiquitin-related proteins that emerged from the genetic screen affect α-syn degradation. However, in strains overexpressing these ubiquitin-related genes we did not detect changes by flow cytometry in steady-state α-syn protein levels (Supplementary Figure 2
). As with our previous analyses (above), the overlap between the data obtained from the genome-wide genetic screen and mRNA profiling assay was minor and statistically insignificant (four genes, p=0.96).
Applying ResponseNet to these disparate datasets revealed a more coherent view of the cellular response (Supplementary Figure 3
). The resulting network provided context to a large portion of the data: 34 (44%) genetic hits and 166 (27%) differentially expressed genes were linked to each other through 106 intermediate connections. These include two thirds of the protein kinase, phosphatase and ubiquitin-related genetic hits, illuminating their intricate role in the response to α-syn. For example, ResponseNet suggests that the genetic suppressor Rck1, a kinase known to respond to oxidative stress, functions through its interactions with the Cad1 transcription factor, and that this sub-network explains the differential transcriptional of seven genes (Supplementary Figure 3J
). Similarly, ResponseNet identifies a set of transcriptional changes that it traces back to the genetic hits Bre5 and Ubp3, which form a deubiquitination complex (Supplementary Figure 3C
The major cellular pathways responding to α-syn toxicity included ubiquitin-dependent protein degradation, cell cycle regulation and vesicle trafficking pathways, all of which have previously been associated with PD (Supplementary text and Supplementary Figure 3
). Impairment of the ubiquitin proteasome system33
and mutations in ubiquitin-related genes (parkin and uch-L1) underlie sporadic and familial forms of PD. Interestingly, parkin is associated with the SCF ubiquitin ligase complex 34
, components of which were selected by ResponseNet. Inappropriate cell cycle regulation has also been implicated in neuronal cell death in PD 35,36
, and ResponseNet predicted several regulators of mitosis and early meiosis. Below we focus on additional ResponseNet predictions that relate to known aspects of PD including nitrosylation, mitochondrial dysfunction and the heat shock response.
Fzf1 was the only gene identified in the screen related to nitrosative stress 37
. However, ResponseNet connected it to four up-regulated transcripts, including Pdi1, a protein disulfide isomerase (PDI) (). Intriguingly, the up-regulation of human PDI protects neuronal cells from neurotoxicity associated with ER stress and protein misfolding (both of which are linked to α-syn expression), and, further, PDI has been found to be S-nitrosylated in PD 38
. We found that increased expression of α-syn causes increased S-nitrosylation of proteins (). This result is surprising as nitrosative responses in yeast cells were long thought to represent a defense mechanism against other microbes. Very recently it was shown that yeast synthesize NO in response to exogenous H2
, suggesting that the nitrosylation of specific proteins is a highly conserved response to oxidative stress.
Nitrosative stress response to α-syn expression in yeast
Mitochondrial dysfunction and oxidative stress have been strongly linked with PD 40
, and were recently associated specifically with α-syn (e.g., 41
). Although mitochondrial dysfunction was a prominent signature in the microarray data (Su et al.; manuscript submitted), the genetic hits contained only a few genes clearly related to mitochondria. ResponseNet identified two connected components related to mitochondrial dysfunction. One component contained the suppressor Hap4, a transcriptional activator of respiratory genes, directly connected to several of the differentially expressed genes (Supplementary Figure 3B
). The other component contained regulators of the retrograde signaling pathway, which senses mitochondrial dysfunction (Mks1, Rtg2 and Grr1 42
, Supplementary Figure 3E
The induction of heat shock response directly or via chemical inhibition of Hsp90 43
suppresses α-syn toxicity in many model systems including yeast, flies, mice and human cells (e.g., 44,45
). However, heat shock related genes were conspicuously absent among the list of genetic suppressors. Nonetheless, ResponseNet predicted the involvement of two highly conserved heat shock regulators, the chaperone Hsp90 (isoform Hsp82, Supplementary Figure 3A
) and the heat shock transcription factor Hsf1 (). Interestingly, ResponseNet predicted that the toxicity suppressor Gip2, a putative regulatory subunit of the Glc7 phosphatase, interacts with Gac1. Gac1 is a regulatory subunit of the Glc7 complex, which is known to activate Hsf146
. This connection suggested that Gip2 overexpression might induce a heat shock response and prompted us to test it. Indeed, we found that strains overexpressing Gip2 show elevated levels of heat shock proteins (). ResponseNet therefore provided a mechanistic explanation for the suppression of α-syn toxicity achieved by Gip2 overexpression and identified a new player in the regulation of the ancient heat shock response.
Overexpression of Gip2 causes induced expression of Hsf1 targets
We also identified cellular pathways whose relation to α-syn toxicity was initially obscure, raising the possibility that they may be interesting avenues for future research. Below we focus on two such highly-conserved pathways, the mevalonate/ergosterol pathway that is targeted by the cholesterol lowering statin drugs, and the target of rapamycin (TOR) pathway.
The mevalonate/ergosterol biosynthesis pathway not only synthesizes sterols, but also synthesizes other products with connections to α-syn toxicity such as farnesyl groups required for vesicle trafficking proteins and ubiquinone required for mitochondrial respiration. ResponseNet ranked highly Hrd1, which regulates the protein target of statins, and the predicted intermediary Hap1, a proposed transcriptional regulator of the pathway 47
(Supplementary Figure 3A
). In addition, the α-syn mRNA profile was modestly correlated with the profile of yeast treated with lovastatin (r=0.32, p< 10−93
, Su et al; manuscript submitted), and several genetic hits could be also associated with products of the pathway (dependent enzymes Bet4 and Cax4, farnesylated proteins Ypt1 and Ykt6 and putative sterol carriers Sut2, Osh2, and Osh3). We therefore tested the effect of lovastatin, which selectively inhibits the highly conserved HMG-CoA reductase of yeast as well as that of mammalian cells, on α-syn toxicity. Addition of 5µM lovastatin to the media caused a further reduction in growth to strains overexpressing α-syn (), but did not reduce growth of either wild-type controls or of cells expressing another toxic protein, a glutamine-expansion variant of huntingtin exon I 48
(Supplementary Figure 4
). We further tested ubiquinone, a downstream output of this pathway, reasoning that its down-regulation through the action of α-syn might increase cellular vulnerability. Indeed, the addition of ubiquinone-2 to the media provided a modest suppression against α-syn toxicity. Ubiquinone is an antioxidant, but this was not a non-specific antioxidant response as the antioxidant N-acetylcysteine had no effect (Supplementary Figure 5
Effects of the small molecules lovastatin and rapamycin on α-syn toxicity
The TOR pathway has been related to other neurodegenerative diseases 49,50
. ResponseNet identified the TOR pathway proteins Tor1, Tor2 and their target transcription factors as intermediary between the genetic hit Lst8 and several up-regulated genes involved in spore wall formation (a vectorially directed secretory process in yeast) and vacuolar protein degradation (). We found that addition of the TOR-inhibitor rapamycin to the media markedly enhanced the toxicity of α-syn. Indeed, a low dose α-syn, which is otherwise innocuous, became toxic (). Establishing the specificity of this effect to α-syn, rapamycin did not reduce growth of cells expressing glutamine expansion variants of huntingtin exon I (Supplementary Figure 6