ABSTRACT
In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans mutants lacking glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.
INTRODUCTION
The maintenance of a functional proteome (also known as proteostasis or protein homeostasis) requires the concerted intervention of different enzymatic and regulatory systems, termed as Proteostasis Network (PN). Thus, PN assists polypeptides to achieve and maintain their active conformation (chaperones and folding factors), provides an adequate cellular environment for their function (unfolded protein response (UPR), heat shock and oxidative responses) and efficiently disposes them when recognized as non-functional or are no longer required (proteasome and autophagy systems) 1, 2. Given its pivotal role in cellular function, deregulation of PN dramatically impacts protein homeostasis and is an underlying cause of several human diseases, collectively known as proteinopathies, mainly characterized by the aberrant deposition of aggregated, misfolded proteins 3. These disorders include some of the most prevalent neurodegenerative diseases like Alzheimer’s, Parkinson’s or Huntington’s Diseases, among others 4. At the core of PN, the pathways involved in reduction, oxidation and isomerization of disulfide bonds are essential to maintain protein homeostasis as they prevent, correct or remove faulty bonds leading to non-functional protein folding.
Protein disulfide bonds are formed at cysteine residues, which can also undergo a variety of other post-translational modifications such as sulfenylation, sulfinylation, nitrosylation or persulfidation, some of which are irreversible 5. The maintenance of protein thiols in their reduced state in the cytosol and mitochondrial matrix is carried out mainly by two dedicated redox pathways: the thioredoxin and glutathione systems6. In contrast, in the lumen of the endoplasmic reticulum (the subcellular compartment where the folding of the proteins entering the secretory pathway takes place), the correct disulfide bond formation is performed by an oxidative folding pathway, largely composed of members of the protein disulfide isomerase (PDI) family 7. Intriguingly, no members of the thioredoxin or glutathione redox systems exist in the endoplasmic reticulum, and the identity of the enzymatic systems providing reducing equivalents to ER-resident PDIs has remained elusive for decades. Recently, the cytoplasmic thioredoxin system has been shown to shuttle electrons into the ER to reduce oxidized PDIs and ensure correct disulfide formation 8. In addition, an active import of cytoplasmic glutathione into ER lumen by specific transporters is key to maintain redox homeostasis in the ER 9, highlighting the importance of a coordinated redox network for maintaining proteostasis across all subcellular compartments.
Organisms as distant as the nematode C. elegans and humans contain a similar number of cysteine residues in their proteome, estimated in about 210,000 10. Importantly, their respective thioredoxin and glutathione redox systems are highly conserved 11, supporting the use of C. elegans as a simple model relevant to addressing key questions in redox biology 12, 13. In worms, the thioredoxin system is dispensable for redox homeostasis as null or strong loss of function mutants of the genes encoding the cytoplasmic and mitochondrial thioredoxins (trx-1, trx-2 and trx-3) and thioredoxin reductases (trxr-1 and trxr-2) are viable and superficially wild type 14, 15, 16, 17. In contrast, mice lacking any member of the cytosolic or mitochondrial thioredoxin system are embryonic lethal (reviewed in 18). However, when bypassing the embryonic requirement by employing conditional knock-outs, the thioredoxin system is also dispensable for viability in mice 18. Thus, in the absence of a functional thioredoxin system, the glutathione system may be responsible for the maintenance of redox homeostasis in both organisms 16, 18, 19. This key role of glutathione is further emphasized by the strict requirement of dietary methionine as a GSH source (generated by the trans-sulfuration pathway) in the absence of functional thioredoxin and glutathione redox systems in mice 19 or the lethal phenotypes of mutations in the genes responsible for glutathione synthesis (gcs-1 and gss-1) or recycling (gsr-1) in worms 10, 20. Furthermore, a study in C. elegans showed that GSH redox potential is highly sensitive to small changes in its oxidation status 10. This sensitivity would translate into rapid responses to even minor perturbations of the cytosolic redox environment and, as consequence, to a quick adjustment of the thiol-disulfide balance of the proteome. Together, these data strongly suggest the glutathione system is the primary enzymatic system responsible for the maintenance of redox homeostasis in metazoa.
Under non-stressed conditions, the cytosol of eukaryotic cells is maintained in a reduced state to favour the stabilization of free thiol groups while the ER environment is more oxidized in order to promote disulfide bond formation 21. Consistent with a tightly controlled interplay between proteostasis and redox homeostasis, proteotoxic stress generated by aggregating proteins in the cytoplasm of both C. elegans and mammalian cells strongly disturb redox homeostasis, causing a shift towards a more oxidizing condition in the cytosol and, conversely, towards a more reducing condition in the ER 22. However, despite the obvious influence of the redox environment in protein folding, whether disruption of redox homeostasis promotes protein aggregation is unknown. Here, we addressed this question by exploring the impact of a compromised glutathione redox environment in C. elegans models of aggregation-prone proteins. We have found that impairment of glutathione homeostasis causes a robust enhancement of phenotypes associated to protein aggregation as a consequence of autophagy disruption and we show this effect is evolutionary conserved in eukaryotes from yeast to mammalian cells.
RESULTS
gsr-1 deficiency exacerbates the phenotypes induced by polyQ expression in C. elegans muscle cells
Although proteotoxic stress may trigger loss of redox homeostasis 22, how impaired redox homeostasis impacts on protein aggregation is unknown. To address this question, we focused on the role of the C. elegans gsr-1 gene, encoding glutathione reductase, for two reasons: Firstly, the glutathione redox system is essential in worms 10, 20 whereas the thioredoxin system is dispensable 14, 15, 16, 17. Secondly, maternal gsr-1 mRNA and/or protein contribution from heterozygous progenitors (balanced with the qC1 balancer 23) allows to work with genetically homozygous gsr-1 animals, designated as gsr-1(m+,z−), because these animals reach adulthood with no discernible phenotype under normal growth conditions 20, in contrast with gss-1 and gcs-1 mutants that are unable to synthesize glutathione and in which maternal contribution only allows growth of the homozygous progeny till L1-L2 larval stages 10.
On this premise, we first downregulated gsr-1 expression by RNAi feeding in worms that constitutively express in muscle cells heterologous aggregation-prone proteins associated to human neurodegenerative diseases such as α-synuclein::YFP (Parkinson) 24, Q40::YFP (polyglutamine diseases like Huntington) 25 and human Aβ (Alzheimer) 26. Despite to the fact that Aβ, a-synuclein and polyQ stretches lack cysteine residues in their sequence, gsr-1 downregulation increased the number of α-synuclein::YFP and Q40::YFP aggregates while it enhanced the paralysis of Aβ worms (a readout of Aβ aggregation) (data now shown). This suggests that the phenotypes caused by gsr-1 downregulation are likely not mediated by direct disulphide bond formation on these proteins. Unexpectedly, worms expressing Q40::YFP in muscle cells displayed a progressive larval arrest phenotype when feeding on gsr-1 RNAi bacteria, an effect that did not occur in transgenic animals expressing the control Q35::YFP fusion protein 25 (Supplementary Figure 1a,b) or in the Aβ and α-synuclein::YFP models (data not shown). Consistent with the RNAi data, gsr-1(m+,z−) animals had a significantly increased number of muscle Q40::YFP aggregates at the L4 (day 3) and young adulthood (day 4) stages (Figure 1a), which inversely correlated with the muscular performance measured by thrashing activity in liquid medium (Supplementary Figure 1c). No effect was found on the aggregation or thrashing phenotypes of rmIs132 [Punc-54::Q35::yfp]; gsr-1(m+,z−) control worms (Figure 1a and data not shown). Transgenic restoration of GSR-1 expression in rmIs133 [Punc-54::Q40::yfp]; gsr-1(m+,z−) worms decreased the number of Q40::YFP aggregates, demonstrating that the increased aggregation phenotype is specific to gsr-1 deficiency and not to a closely linked unknown mutation (Supplementary Figure 1d). Furthermore, pharmacological inhibition of GSH synthesis (by buthionine sulfoximine, BSO), GSSG reduction (by 1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU) and GSH depletion (by diethyl maleate, DEM) phenocopied the developmental delay phenotype caused by gsr-1 downregulation in worms expressing Q40::YFP fusion protein (Supplementary Figure 1e,f and Figure 1b). Remarkably, DEM had a much more profound effect than BSO or BCNU as rmIs133 [Punc-54::Q40::yfp] hermaphrodites exposed to 2.5 mM DEM rapidly became sick and laid very few progeny which invariably arrested at L1-L2 larval stage (Figure 1b). Together, these data demonstrate that reduced GSR-1 and GSH levels compromise muscle function and survival of worms expressing Q40::YFP in muscle cells.
gsr-1(m−,z−) embryos expressing Q40::YFP in muscle cells undergo dramatic cell blebbing and exploding phenotypes
We previously reported that gsr-1(m−,z−) embryos, lacking maternal contribution, arrest at the pregastrula/gastrula stage with the embryonic cells superficially normal and no apparent signs of necrosis or apoptosis (Figure 1c and Movie 1) 20. In turn, rmIs133 [Punc-54::Q40::yfp] embryos develop normally until hatching, similar to wild type embryos (Figure 1d and Movie 2) 25. Surprisingly we identified a robust synthetic phenotype in rmIs133 [Punc-54::Q40::yfp]; gsr-1(m−,z−) embryos as most of these embryos undergo dramatic cell blebbing and/or sudden catastrophic embryo explosion phenotypes when maintained in water at 25°C (Figure 1e,f, Movies 3-4 and Table 1). These phenotypes also happened, although to a lesser extent, in gsr-1(m−,z−) embryos expressing a Q35::YFP fusion protein from the rmIs132 [Punc-54::Q35::yfp] transgene (Table 1). Importantly, when these embryos are maintained in an isotonic buffer, they do not explode but maintain the blebbing phenotype, suggesting the eggshell integrity, which preserves embryonic osmotic homeostasis, is compromised in gsr-1(m−,z−) embryos expressing Q35::YFP or Q40::YFP proteins (Table 1).
Under normal conditions, the plasma membrane of eukaryotic cells is tightly bound to an underlying actomyosin cortex that contributes to cell shape maintenance 27. However, when the actomyosin cortex integrity is disrupted, actin-devoid evaginations (blebs) are generated by internal hydrostatic pressure 28. To test whether impairment of the actomyosin cortex function might underlay the blebbing phenotype of the rmIs133 [Punc-54::Q40::yfp]; gsr-1(m−,z−) embryos, we examined the dynamics of actin fluorescence markers. The distribution of actin filaments in rmIs133; gsr-1(m−,z−) embryos did not markedly differ from that of rmIs133; gsr-1(m+,z+) controls and actin was also detected at the sites of blebs (Figure 1g and Movie 5). As microtubule network destabilization can also result in cell blebbing 29, 30, we next asked whether tubulin dynamics was altered in rmIs133; gsr-1(m−,z−) embryos. Using tubulin fluorescence markers, we identified an aberrant distribution of the tubulin filaments in the periphery of the rmIs133; gsr-1(m−,z−) embryonic cells that is not observed in control embryos (Figure 1h and Movie 6). Similar results were obtained with tubulin immunostaining (Supplementary Figure 2). These data suggest that, in a GSH compromised environment, polyQ proteins modify the dynamics of the microtubule network, impinging on plasma membrane integrity and function.
gsr-1 mutants show enhanced polyQ proteins aggregation in neurons and intestinal cells
To determine whether the protective effect of GSR-1 in polyQ worms may be restricted to muscle cells or extended to other tissues, we analyzed gsr-1(m+,z−) animals expressing polyQ proteins in neuronal and intestinal cells. In addition, to pharmacologically validate the data obtained with gsr-1(m+,z−) mutants in these models, we opted for depleting the pool of reduced glutathione by DEM treatment as it produced the more penetrant phenotype on rmIs133 [Punc-54::Q40::yfp] worms (Figure 1b). First, we used a C. elegans model of Machado-Joseph Disease pathogenesis in which mutant human Ataxin-3 (ATXN3) fused to YFP is expressed in a pan-neuronal fashion 31. Similar to muscular polyQ models, we found that the gsr-1(m+,z−) mutation caused a robust increase of ventral nerve cord fluorescent foci in worms expressing mutant ATXN3::Q130::YFP but not in ATXN3::Q75::YFP worms that served as non-aggregating controls (Figure 2a). However, upon exposure of animals to DEM, we observed an inverse correlation with toxicity as the ATXN3::Q75::YFP control worms suffered a significant developmental delay while, in contrast, ATXN3::Q130::YFP worms developed at the same rate compared to untreated controls (Figure 2b). These results suggest that a protective mechanism might operate in neurons when a certain aggregation threshold is surpassed.
We further confirmed the deleterious effect of the gsr-1 mutation on neuronal polyQ toxicity using worms that express in the six touch receptor neurons the first 57 amino acids of the human huntingtin protein followed by normal (Q19) or expanded (Q128) polyglutamines and that is fused to CFP 32. While the gsr-1 mutation did not modify light touch response of worms expressing the HTT57::Q19::CFP fusion protein, we observed a significant decrease of the touch response in HTT57::Q128::CFP nematodes (Figure 2c). In contrast to the ataxin model, we did not identify any differential growth response of HTT57::Q19::CFP and HTT57::Q28::CFP worms to DEM treatment (Figure 2d), most likely due to the dispensability of touch neurons for development.
Finally, we also assayed the impact of the gsr-1 mutation in worms expressing a Q44::YFP fusion protein in intestinal cells 33. At 20°C, the appearance onset of intestinal fluorescent aggregates was similar in both wild type and gsr-1(m+,z−) backgrounds (Figure 2e). However, at 16°C, the formation of intestinal Q44::YFP aggregates occurred earlier and these aggregates were bigger and more abundant in gsr-1(m+,z−) worms compared to wild type controls (Figure 2e). In consonance, animals expressing Q44::YFP proteins were highly sensitive to DEM (Figure 2f). Collectively, these data indicate that gsr-1 and GSH deficiency enhance polyQ proteins aggregation regardless of the cell type or tissue of expression.
gsr-1 deficiency is deleterious in C. elegans expressing human Aβ and α-synuclein proteins
In addition to the effect on polyQ proteins aggregation, the previously mentioned enhanced paralysis of worms expressing human Aβ or increased α-synuclein::YFP aggregation in animals fed with gsr-1 RNAi pointed to a general effect of gsr-1 in proteostasis. We then characterized the effect of the gsr-1 mutation in these two additional models of protein aggregation. Worms expressing the dvIs2 [Punc-54::Aβ] transgene produce human Aβ protein in muscle cells where it aggregates and causes age-dependent progressive paralysis at 20°C but not (or very mildly) at the 16°C permissive temperature 26. As shown in Figure 3a, the gsr-1(m+,z−) mutation induced robust paralysis in dvIs2 [Punc-54::Aβ] worms at the permissive temperature. This synthetic interaction was phenocopied pharmacologically with DEM, causing a complete L1-L2 larval arrest phenotype of dvIs2 worms (Figure 3b). Moreover, dvIs2 [Punc-54::Aβ]; gsr-1(m−,z−) embryos also showed blebbing and explosion phenotypes (Table 1), similar to gsr-1(m−,z−) embryos expressing Q40::YFP under the control of the muscle specific unc-54 promoter (Figure 1e,f). Interestingly, an even more striking phenotype was found when combining the gsr-1(m+,z−) mutation with a different Aβ transgene, dvIs14 [Punc-54::Aβ] 34. In this case, dvIs14; gsr-1(m+,z−) worms at the permissive temperature displayed a much faster paralysis as compared to that of dvIs2; gsr-1(m+,z−) counterparts (Figure 3c). In addition, dvIs14; gsr-1(m+,z−) animals showed a fully penetrant egg-laying phenotype, most likely reflecting the lack of contractility of the uterine and vulva muscles which are responsible for egg extrusion (Figure 3d). The genetic interaction between Aβ protein and the gsr-1 mutation was also found in worms expressing human Aβ in the nervous system from the ganIs2 [Punc-119::Aβ] transgene 35. Thus, the gsr-1(m+,z−) mutation causes a strong developmental delay in a ganIs2 genetic background (Figure 3e) which correlated with a higher sensitivity to DEM of ganIs2 nematodes (Figure 3f).
Likewise, the aggregation in muscle cells of the α-synuclein::YFP fusion protein expressed from the pkIs2386 [Punc-54::a-syn::yfp] transgene 24 was strongly enhanced in gsr-1(m+,z−) mutants (Figure 4a) although, in contrast to animals expressing Q40::YFP or Aβ in muscle cells, DEM treatment did not cause any developmental phenotype in muscle α-synuclein::YFP animals (Figure 4b). Similarly, in baIn11 [Pdat-1::α-synuclein; Pdat-1::gfp] animals expressing α-synuclein in the six worm head dopaminergic neurons 36, the gsr-1(m+,z−) mutation significantly increased neurodegeneration in an age-dependent manner (Figure 4c) whereas no phenotype was observed when these animals are treated with DEM (Figure 4d). The lack of any deleterious effect of DEM treatment in worms expressing α-synuclein::YFP in muscle cells may be due to the very mild effect on motility which has been reported for these worms 24 in comparison to human Aβ or Q40::YFP expressing animals 25, 26. Similarly, the dispensability of dopaminergic signalling for worm survival 37 may explain the absence of deleterious phenotypes of worms expressing α-synuclein in dopaminergic neurons when exposed to DEM.
gsr-1 deficiency worsens the phenotypes of C. elegans expressing endogenous metastable proteins
All models so far described in this work employ heterologous aggregation-prone proteins (Aβ, α-synuclein::GFP or polyQ::GFP) not naturally found in C. elegans. To rule out that the effect of the gsr-1(m+,z−) mutation in these models might be a consequence of expressing foreign proteins in worm cells, we monitored mutant phenotypes in animals bearing thermosensitive alleles of endogenous genes, which encode metastable proteins that are highly sensitive to changes in the protein folding environment. To this end, we used unc-52(e669su250) and let-60(ga89) mutants that display a highly penetrant paralysis or embryonic arrest phenotype, respectively, at the non-permissive temperature of 25°C but are roughly wild type at the permissive temperature of 16°C 38. Consistent with the results above, the double mutant unc-52(e669su250); gsr-1(m+,z−) displayed a robust paralysis phenotype at 16°C (Figure 4e). This synthetic interaction was phenocopied by treatment of unc-52(e669su250) mutants with 2mM DEM, causing a fully penetrant early larval arrest (Figure 4f). Likewise, a strong synthetic interaction was found between let-60(ga89) and gsr-1 mutants as shown by the impossibility to generate viable progeny from let-60(ga89); gsr-1/qC1::rfp balanced animals at permissive temperature (Figure 4g). To rule out a possible deleterious effect of the qC1::rfp balancer, we stabilized the gsr-1(tm3574) mutation with a different version of the qC1 balancer tagged with GFP (see Supplementary Table 1 for full genomic information of both balancers) that resulted in 50% viable progeny (Figure 4h). Remarkably, none of this viable progeny was gsr-1(m+,z−), but all gsr-1(m+,z+), thus confirming the synthetic lethal phenotype of gsr-1(m+,z−) and let-60(ga89) mutants which was further supported by the larval arrest phenotype of let-60(ga89) animals in the presence of 3mM DEM (Figure 4i). Together, these data suggest that glutathione reductase and the maintenance of a proper GSH redox homeostasis may protect against protein aggregation.
Autophagy function is impaired in gsr-1 mutants
Proteostasis is maintained by a delicate equilibrium of different molecular pathways. Some of these pathways are at the core of the proteome maintenance such as protein synthesis, folding, trafficking or degradation while others act as modifiers of proteostasis such as unfolded protein response, nutritional and metabolic status, stress situations, genetic and epigenetic susceptibility or physiological processes like aging 2. To ascertain which of these pathways might underlay the protective effect of GSR-1 and GSH in protein aggregation, we first tested the effect of gsr-1 deficiency on different reporters of proteostasis regulators (including insulin, proteasome, UPR-ER, heat-shock, mitophagy or autophagy pathways), under basal and stress conditions.
As shown in Supplementary Figure 3, the gsr-1(m+,z−) mutation only caused a mild downregulation of the UPR-ER reporter HSP-4::GFP under both conditions (Supplementary Figure 3c) and an induction of the oxidative stress reporter SOD-3::GFP under basal, but not stressed conditions (Supplementary Figure 3b). As SOD-3::GFP induction was not accompanied by an increase in DAF-16::GFP nuclear translocation (Supplementary Figure 3a), the effect of the gsr-1 mutation on sod-3 transcription is more likely due to a DAF-16 independent response to oxidative stress rather than a role in proteostasis.
In contrast to all reporters described in Supplementary Figure 3, we identified a clear effect of the gsr-1 mutation in worms carrying the sqIs17 transgene, that expresses the HLH-30::GFP autophagy marker 39. HLH-30 is the worm orthologue of the mammalian TFEB transcription factor and, under normal growth conditions, these transcription factors are inactive and diffusely distributed in the cytoplasm. However, upon stress conditions such as starvation and heat-shock, HLH-30/TFEB translocate into the nucleus, triggering the transcription of genes required for autophagy induction and lysosomal biogenesis 39, 40, 41, 42. We found that the cytosolic subcellular localization of HLH-30::GFP was not modified in a gsr-1(m+,z−) mutant background in well-fed animals (Figure 5a). In turn, one hour starvation or 30°C heat-shock was enough to induce a strong nuclear translocation of HLH-30::GFP, a phenomenon severely hampered in gsr-1(m+,z−) worms subjected to starvation, but not heat-shock (Figure 5a and Supplementary Figure 4a). These results suggest that gsr-1 deficiency may negatively impact on proteostasis by impairing autophagy-dependent protein degradation, at least upon specific stimuli. To test for this possibility, we used worms that carry the bpIs151 transgene and express SQST-1::GFP, a reporter protein that is typically degraded by autophagy in non-stressed conditions and that accumulates in the form of fluorescent foci, mainly in embryonic and larval intestinal cells, when autophagy is compromised 43. Consistently, we found no SQST-1::GFP foci in bpIs151; gsr-1(m+,z−) embryos and L1 larvae under normal growth conditions (Figure 5b,c). However, 24 hours starvation or a mild impairment of autophagy by RNAi downregulation of lgg-1/LC3 (required for autophagosome biogenesis) increased the number of SQST-1::GFP foci in gsr-1(m+,z−) embryos and L1 larvae, respectively, as compared to wild type controls (Figure 5b,c and Supplementary Figure 4b). These results identify GSR-1 as a novel positive regulator of autophagy.
Next, we reasoned that if gsr-1 mutants enhance the phenotypes of worm models of proteostasis by impairing autophagy, then genetically blocking autophagy in these proteostasis models would similarly result in an enhancement of their deleterious phenotypes. To our knowledge, no reports have so far addressed the implication of autophagy in the phenotypes of unc-52 or let-60 thermosensitive mutants while a role for autophagy on the phenotypes of worms expressing human Aβ, α-synuclein and polyQ proteins has only been studied by RNAi downregulation 42, 44, 45, 46. To provide more compelling evidence of the implication of autophagy in these proteostasis models, we employed atg-3(bp412) mutants, in which autophagy is blocked at the autophagosome formation step 43. We corroborated the RNAi results in the muscle Aβ, α-synuclein and polyQ worm models and found an enhancement of their respective paralysis or aggregation phenotypes in an atg-3 background (Figure 5d-f). Interestingly, we also found a significant enhancement of the paralysis in unc-52; atg-3 double mutants compared to unc-52 controls at permissive temperature (Figure 5g), a result that was phenocopied using lgg-1 RNAi (Supplementary Figure 4c). We also attempted to evaluate the effect of the atg-3(bp412) mutation in the embryonic arrest phenotype of let-60(ga89) mutants but we failed to isolate a double mutant atg-3; let-60, which possibly indicates a lethal interaction. In support of this possibility, let-60(ga89) mutants expressing the autophagy substrate SQST-1::GFP display a ten-fold increase in the embryonic arrest phenotype at the permissive temperature (Figure 5h). Together, these data reveal that the endogenous, aggregation-prone metastable UNC-52 and LET-60 proteins are likely degraded by autophagy and reinforce the implication of autophagy in dismissing heterologous Aβ, α-synuclein and polyQ proteins in worm models of neurodegenerative diseases.
Glutathione depletion has been shown to increase protein carbonylation and aggregation 47. We hypothesized that the deleterious effect of the gsr-1 mutation on proteostasis could be explained by an increase of the load of proteins to be degraded by autophagy, ultimately resulting in a severe impairment of the autophagy machinery. To test this hypothesis, we aimed to generate atg-3(bp412) and gsr-1(m+,z−) double mutants in the different protein-aggregation models and we faced strong synthetic interactions. For instance, we were unable to generate viable atg-3; gsr-1(m+,z−) animals expressing human Aβ protein in muscle cells (either from dvIs2 or dvIs14 transgenes). In consonance, although viable, atg-3; gsr-1(m+,z−) worms expressing a-SYN::GFP in muscle cells were sick and displayed a high percentage of internal hatching, which precluded proper quantification of paralysis (data not shown). Likewise, atg-3; gsr-1(m+,z−) animals expressing Q40::YFP in muscle cells were viable but had increased embryonic arrest phenotype (Supplementary Figure 4d). Only the triple atg-3; gsr-1(m+,z−); unc-52 failed to show an increased paralysis phenotype compared to the two control strains atg-3; unc-52 and gsr-1(m+,z−); unc-52 (Figure 5g). However, increasing the autophagy load by expressing the SQST-1::GFP autophagy substrate in unc-52; gsr-1(m+,z−) worms resulted in a much earlier paralysis onset compared to unc-52; gsr-1(m+,z−) or unc-52; bpIs151 double mutant controls (Figure 5i). Furthermore, a triple atg-3; gsr-1/qC1::rfp; bpIs151 produced very few gsr-1(m+,z−) progeny which were very sick and arrested early during development (data not shown). Similarly, we were also unable to isolate triples let-60; atg-3; bpIs151 or let-60; gsr-1(m+,z−); bpIs151, reinforcing the idea that metastable LET-60 is also degraded by autophagy. Together, the fact that gsr-1(m+,z−) mutants strongly enhance the deleterious phenotypes caused by aggregation-prone proteins in worms with compromised autophagy function (i.e. atg-3 mutations or by expression SQST-1::GFP protein) pinpoints a key role of GSR-1 in maintaining a healthy proteome.
Glutathione reductase mutation and glutathione depletion increase protein aggregation in yeast and mammalian cell cultures
To determine whether the protective role of the glutathione system on protein aggregation and associated phenotypes might be restricted to C. elegans or instead is a more universal, evolutionarily conserved mechanism, we turned to a Saccharomyces cerevisiae model of polyQ toxicity48. Importantly, the mechanism by which glutathione reductase regulates polyQ aggregation appears to be maintained as yeast cells lacking glutathione reductase (carrying a Δglr1 deletion) and expressing the aggregation-prone Q103-GFP fusion protein displayed a clear aggregation pattern, not detected in the control strain expressing a non-aggregating Q25-GFP fusion protein (Figure 6a). Noteworthy, the aggregation phenotype of Q103-GFP expressing cells was much more evident when yeast have reached the diauxic phase (Figure 6a), when cells shift from a glucose fermentative to an ethanol respiration mitochondrial oxidative metabolism. Interestingly, protein aggregation of Q103-GFP was increased in Δglr1 cells compared to a WT background only at diauxic phase, when oxidative stress generated by the metabolic shift occurred. In addition, Q103-gfp yeast had a significant growth delay in a Δglr1 background and were more sensitive to DEM treatment than control Q25-GFP expressing cells (Figure 6b-c), reminiscent of the larval developmental arrest phenotype described for Q40::YFP worms with downregulated gsr-1 levels (Supplemental Figure 1a,b) or exposed to DEM (Figure 1b). To test whether autophagy is also involved in maintaining yeast proteostasis when redox homeostasis is challenged, we generated a double Δglr1Δatg8 mutant in Q25-gfp and Q103-gfp backgrounds. atg8 encodes an ubiquitin-like protein which is essential for autophagosome formation 49. As shown in Figure 6d, single Δglr1 or Δatg8 mutants grew similar to wild type controls independently of expressing Q25-GFP or Q103-GFP proteins. However, a mild synthetic growth defect was detected in the double Δglr1Δatg8 in both Q25-gfp and Q103-gfp backgrounds (Figure 6d) and this synthetic interaction was further enhanced when cells were exposed to 2.5 mM DEM (Figure 6e). These data suggest that impairment of glutathione redox homeostasis negatively impacts on proteostasis in yeast cells expressing aggregation-prone proteins, involving autophagy as a mechanism to dismiss the increased load of protein aggregation.
Next, we addressed the protective effect of GSH homeostasis in a mammalian system using the SH-SY5Y neuroblastoma cell line stably transfected with the human APP protein, which drives intracellular accumulation of human Aβ 50. Interestingly, when exposing these cells to sublethal doses of BSO and DEM (Supplementary Figure 5), we found that both treatments promoted an increase of Aβ labelling in the cytoplasm of these cells (Figure 6f). Moreover, the number of cells with Aβ foci that colocalize with thioflavin, which labels amyloid fibrils and aggregates, was strongly induced upon BSO and DEM treatments (Figure 6g). Given that SH-SY5Y cells overexpressing APP have enhanced autophagy 50, the increased Aβ aggregation observed in BSO and DEM treated cells is consistent with an impairment of autophagy.
Collectively, these data confirm that the deleterious effect of GSH and glutathione reductase deficiency on protein aggregation may be conserved from lower eukaryotes to mammals, impinging on the autophagy machinery.
Discussion
The maintenance of a functional proteome is essential for organismal survival and, among the molecular pathways that modulate proteostasis, redox regulation arises as a main regulatory mechanism allowing proteins to attain their native conformation and functionality. It has recently been proposed that the glutathione couple GSH/GSSG, rather than operating as a redox buffer would instead act as a surveillance relay to detect changes of the intracellular redox environment to tightly control the thiol-disulfide balance of the cellular proteome 10. In this study, we present a set of experiments that support this premise of the glutathione system being essential to preserve a healthy proteome, showing that genetic or pharmacological disruption of glutathione redox homeostasis enhances protein aggregation by a mechanism that impinges on the efficacy of autophagy.
Loss of glutathione redox homeostasis enhances the toxicity of protein aggregation
Pharmacological inhibition of glutathione synthesis and recycling as well as glutathione depletion phenocopies the effect of the gsr-1 mutation in worms expressing aggregation-prone proteins. Surprisingly, treatment with DEM in C. elegans expressing Aβ, Q40::YFP and metastable UNC-52 proteins in muscle cells caused a fully penetrant lethality phenotype. DEM is an alkylating agent that has been traditionally used to deplete GSH but a recent study has shown that DEM depletes not only GSH but also protein thiols51. Hence, it is plausible that DEM causes an overload of oxidatively compromised proteins, possibly deemed for autophagic degradation, in addition to its direct effect on the GSH/GSSG ratio. This double impact may underlie the enhanced phenotypes observed in the worm proteostasis models exposed to DEM as compared to those carrying the gsr-1 mutation. Although it has been proposed that cytotoxicity can be genetically uncoupled from aggregation 52, we found a clear correlation of increased aggregation and DEM toxicity when aggregation-prone proteins are expressed in muscle cells. However, this correlation was not so strong for proteins aggregating in neurons. We indeed found one case in which cytotoxicity inversely correlates with aggregation, namely in worms that express the human ATXN3::Q130::YFP protein in neurons and that are highly resistant to DEM treatment despite undergoing extensive aggregation, contrasting with worms that express non-aggregating ATXN3::Q75::YFP protein which are very sensitive to DEM (Figure 2a,b). The resistance of ATXN3::Q130::YFP worms to the effects of DEM could be explained by an increase of exophers production, an extrusion mechanism mediated by very large extracellular vesicles containing damaged material such as aggregated proteins and that is enhanced when mitochondria are compromised 53, as is the case of gsr-1 mutants 20. Plasma membrane blebbing can also be observed in body wall muscle cells of transgenic worms expressing human Aβ, and these blebs contain Aβ aggregates (CD Link, unpublished data). Therefore, body wall muscle cells might also generate exophers under specific conditions and tissue-specific penetrance of the gsr-1 mutation or sensitivity to DEM treatment on exophers formation may account for the differential sensitivity of neuron versus muscle worm proteostasis models.
An unexpected phenotype identified in our experiments is the cell blebbing and explosion of gsr-1(m−,z−) embryos that carry the rmIs133 [Punc-54::Q40::YFP] or dvIs2 [Punc-54::human Aβ] transgenes. To our knowledge, such striking phenotypes have not been previously described in C. elegans embryos. Cell blebs arise from transient disruption of the actomyosin cortex underneath the plasma membrane 28 and we were surprised to find that actin network distribution did not differ obviously from that of non-blebbing controls. In turn, we found that microtubules were aberrantly distributed in the periphery of the gsr-1(m−,z−); rmls133[Punc-54::Q40::YFP] embryonic cells. A recent report has identified the worm calponin CHDP-1 protein as promoter of cell protrusions in C. elegans. CHDP-1 is located at the cell cortex but, surprisingly, does not bind actin 54. Instead CHDP-1 associates with the Rho small GTPase family member Rac1/CED-10 which, among other functions, mediates microtubule dynamics and lamellipodial protrusions 55, 56, 57. Interestingly, we have found that ced-10(t1875) null mutant embryos also undergo cell blebbing, although these blebs are not as large and prominent as those observed in gsr-1(m−,z−); rmIs133 embryos (Movie 7). Furthermore, ced-10 mutants (both reduction and gain of function) increase α-synuclein::GFP and Q40::YFP aggregates in worm muscle cells and enhance α-synuclein dependent dopaminergic neurodegeneration (58 and A. Miranda-Vizuete, unpublished data), suggesting that CED-10 could mediate the protective effect of GSR-1 in these models. How could GSR-1 control CED-10 function? One possibility is via glutathionylation (a post-translational modification whereby a glutathione moiety oxidizes a specific cysteine residue 59), as mammalian Rac1 is glutathionylated at Cys18, a residue conserved in worm CED-10 protein, resulting in increased lamellipodia formation 60. Given that glutathionylation is carried out by glutaredoxins, a class of small redox proteins that are reduced by GSH 61, it will be very interesting to explore whether glutaredoxins mediate the protective effect of GSR-1 in proteostasis maintenance.
Role of cytoplasmic versus mitochondrial glutathione reductase in proteostasis
Like the majority of eukaryotes, C. elegans uses the flavoenzyme glutathione reductase GSR-1 to recycle reduced glutathione from its oxidized form. In worms, gsr-1 is an essential gene that encodes two different GSR-1 isoforms, one located in the cytosol and the other in mitochondria. While GSR-1 activity in the cytoplasm is absolutely required for embryonic development, consistent with glutathione synthesis occurring in this compartment, mitochondrial GSR-1 is dispensable under nonstressed conditions (20 and A. Miranda-Vizuete unpublished data). Surprisingly, gsr-1(m+,z−) animals exhibit mitochondria associated phenotypes such as mitochondrial fragmentation, induction of mitochondrial UPR and lowered mitochondrial DNA content 20. All the proteostasis models used in our study generate aggregated proteins in the cytosol, suggesting that the absence of GSR-1 activity in this compartment is responsible for the synthetic phenotypes observed in gsr-1(m+,z−) mutants. However, we cannot rule out a possible role of the mitochondrial GSR-1 isoform as mitochondria have been shown to import and degrade aggregated proteins originally produced in the cytoplasm 62, 63. Because gsr-1(m+,z−) mutants display phenotypes associated to mitochondrial dysfunction, which might interfere with the import and degradation of the aggregated cytoplasmic proteins into this organelle, the implication of mitochondrial GSR-1 in proteostasis needs to be evaluated separately from the cytoplasmic isoform. Moreover, yeast Q103-GFP aggregates are more prominent in the diauxic phase, in which cells shift from a cytosolic fermentative to a mitochondrial respiratory metabolism, further supporting the possibility that mitochondrial glutathione reductase may have a role in protein aggregation.
Glutathione-dependent regulation of autophagy
Autophagy is an evolutionarily conserved degradation system of defective proteins and subcellular structures, and an early event in autophagy induction is the nuclear translocation and transactivation of the basic Helix-Loop-Helix transcription factor HLH-30 39, 38. Oxidative stress is one of the various conditions that activate autophagy (reviewed in 64, 65) and several components of the autophagy machinery are redox regulated by ROS-mediated mechanisms in different organisms 66, 67, 68. Using a HyPer fluorescent biosensor 69 we found that gsr-1(m+,z−) worms have wild type levels of H2O2 (A. Miranda-Vizuete, unpublished results), suggesting that they are not under oxidative stress and consistent with the lack of HLH-30::GFP nuclear translocation or increased SQST-1:GFP foci in these animals. Similarly, the gsr-1 mutation does not interfere with HLH-30 nuclear translocation upon heat-shock, another ROS-producing treatment that induces autophagy 42,70. In turn, gsr-1(m+,z−) mutants prevent HLH-30 nuclear translocation and degradation of the autophagy substrate p62/SQST-1 upon starvation, a condition that induces autophagy mainly through inhibition of the mTOR pathway 71. Together, these data suggest that the regulation of HLH-30 subcellular localization by GSR-1 is stimulus-specific, likely mediated by mechanisms that do not involve ROS signaling. Strikingly, C. elegans HLH-30 and mammalian TFEB proteins possess only one cysteine residue, located within (C. elegans) or in close proximity (mammalian) to their respective bHLH domains (Supplementary Figure 6). Similar to CED-10 regulation discussed above, glutathionylation of this only cysteine residue of HLH-30 could be a possible mechanism by which GSR-1 regulates HLH-30 nuclear translocation and transactivation, as it has been described for other transcription factors 61, 72, 73.
We previously showed that gsr-1 mutants induce SKN-1 regulated genes of the glutathione pathway like gcs-1 and gst-4 20. Moreover, GSR-1 levels are strongly upregulated upon starvation by a mechanism dependent on the SKN-1 transcription factor 74. Although SKN-1 may regulate mitophagy, a form of autophagy that specifically degrades defective mitochondria 75, it is not clear whether it can also regulate autophagy and protein aggregation in worms. Nonetheless, the Nrf2 transcription factor, which is the SKN-1 mammalian orthologue, is strongly associated to autophagy and proteotoxicity 76, 77, thus supporting the possibility that SKN-1 is a key player in the molecular mechanisms underlying GSR-1 function in these scenarios. Indeed, TFEB and Nrf2 transcription factors may cooperate to stimulate autophagy in mammalian cells78, making likely this cooperation may also happen in C. elegans for increasing GSR-1 levels upon starvation.
Additional evidence of GSR-1 impinging on autophagy comes from the fact that gsr-1(m+,z−) mutants further enhance the deleterious phenotypes caused by aggregation-prone proteins in worms with compromised autophagy function either by atg-3 mutations or by overexpression of the SQST-1::GFP protein, which is specifically degraded by autophagy. This is nicely illustrated in unc-52; gsr-1(m+,z−); sqst-1::gfp worms, as they develop a much earlier onset of paralysis than the respective controls, probably by prioritizing SQST-1::GFP degradation and thus increasing the amount of aggregating UNC-52 that accelerates paralysis. Altogether, the synthetic phenotypes described in this study can be explained by a synergistic overload of misfolded proteins that cannot be degraded by autophagy, which ultimately surpasses a threshold over which cell function is severely affected eventually causing organismal death.
In summary, we found the glutathione system may be a major regulator of protein homeostasis as observed in C. elegans, yeast and mammalian cell models. Our data provide a new conceptual and experimental model to address the molecular mechanisms underlying redox regulation of proteostasis. Given the evolutionary conservation of this function, we postulate that strategies aimed to maintain glutathione redox homeostasis may have a therapeutic potential in diseases associated with protein aggregation, such as most prevalent neurodegenerative diseases.
MATERIALS AND METHODS
C. elegans strains
The standard methods used for culturing and maintenance of C. elegans were as previously described 79. A list of all strains and plasmids used and generated in this study is provided in Supplementary Table 1. All VZ strains are 6x backcrossed with N2 wild type. All transgenes used in this study are stably expressed from genomic integrated lines, except for strains carrying the vzEx169 and vzEx170 extrachromosomal arrays. Details on PCR, sequencing or restriction enzyme genotyping of the different alleles as well as plasmid constructs used in this work can be provided upon request. Unless otherwise noted, all experiments were performed on synchronized worms generated by allowing 10 to 15 gravid hermaphrodites to lay eggs during two to three hours on seeded plates at 20°C.
S. cerevisiae strains
A list of all yeast strains and plasmids used and generated in this study is provided in Supplementary Table 2. Yeast transformation was performed using a standard lithium acetate/polyethylene glycol method and all our experimentation used freshly transformed cells.
Mammalian cell culture
Human SH-SY5Y cells were routinely grown at 37°C in a humidified incubator with 5% CO2 in DMEM F12+Glutamax, supplemented with 10% fetal bovine serum. All experiments were performed after 24 h of incubation. To explore whether BSO and DEM were able to induce intracellular aggregates of amyloid-beta, cells were exposed to BSO (75 mM) and DEM (25 μM) for 18 h.
Glutathione pathway inhibitors
Appropriate volumes from stock solutions of BSO (Sigma B2515, 200 mM in H2O), BCNU (Sigma C0400, 20 mg/ml EtOH) and DEM (Sigma D97703, directly from the supplier, diluted in 0,5% DMSO final concentration on plates) were added to NGM prior pouring the plates. Plates were freshly prepared to avoid degradation of the chemicals with time. The developmental stages were scored in a SMZ645 Nikon stereoscope at specified days (depending on the growth temperature and genotype) after synchronized egg-lay.
Quantification of polyQ C. elegans strains phenotypes
polyQ::YFP aggregates quantification
For worms expressing the rmIs132 [Punc-54::Q35::yfp] and rmIs133 [Punc-54::Q40::yfp] transgenes, muscle YFP aggregates were scored in the whole animal. Neuronal YFP aggregates in worms expressing the rmIs237 [Prgef-1::AT3v1-1::Q75::yfp] and rmIs263 [Prgef-1::AT3v1-1::Q130::yfp] transgenes were scored in the ventral nerve cord segment from the pharynx to the vulva. For worms expressing the drIs20 [Pvha-6::Q44::yfp] transgene, we scored the number of animals with 10 or more distinct YFP aggregates in the intestinal cells. All YFP aggregates quantifications were performed at 40x (muscle or intestinal aggregates) or 100x (neuronal aggregates) magnification in an Olympus BX61 fluorescence microscope equipped with a DP72 digital camera coupled to CellSens Software for image acquisition and analysis.
Mechanosensory response
The mechanosensory function in worms expressing the igIs245 [Pmec-3::htt57-19Q::cfp] and igIs1 [Pmec-3::htt57-128Q::cfp] transgenes was assayed by the touch response assay 80. Briefly, first day adult worms were gently touched on their tail with an eyelash mounted on a toothpick and their forward movement response was scored by 10 touches per worm assayed.
Time-lapse recording of blebbing and exploding embryo phenotypes
For the DIC recordings embryos were prepared and mounted as described 81. Gravid hermaphrodites were dissected, and two-to four-cell-stage embryos were mounted on 4% agar pads in water and sealed with Vaseline. 4D-microscopy (multifocal time lapse microscopy) was carried out using a Leica DM6000 Microscope fitted with Nomarski optics. Recordings were made using a 100X/1.4 PL APO objective, and the temperature was kept constant at 25°C. The microscope was controlled with the open-source software Micro-manager. Images from 30 focal planes (1micron/section) were taken every 30 seconds for up to 15 hours. Images and videos were processed with the Xnview and Quicktime software. For fluorescence recordings, animals were grown at 16°C and shifted to 25°C approximately 24h prior to observation. Gravid hermaphrodites were dissected and gsr-1(m+,z+) and gsr-1(m−,z−) embryos were carefully placed side by side on a ~2% agarose pad in either H2O (Figure 1g) or M9 (Figure 1h). A coverslip was placed on top of the embryos and sealed with VALAP 82. Confocal images were acquired on a Nikon A1R microscope through a 60x/1.4 PlanApo objective using a pinhole of 1.2 Airy Units (39.7 μm); 3-5 focal planes separated by 2-3 μm were recorded every 3-10 minutes for a total of 16 h at 22-24°C. For mounting videos, a single focal plane was selected for each time point.
Quantification of β-amyloid C. elegans strains phenotypes
Paralysis
Worms expressing the dvIs2 [Punc-54::human Aβ] or dvIs14 [Punc-54::human Aβ] transgenes (and respective controls) were grown at 16°C and paralysis scoring was initiated four days after egg-lay and determined daily, whereby paralyzed worms were removed from plates. A worm was scored as paralyzed if it did not respond to a gentle touch stimulus with a platinum wire.
Internal hatching
Animals carrying the dvIs14 [Punc-54::human Aβ] transgene were grown at 16°C and the internal hatching phenotype was determined after adulthood by scoring their inability to extrude embryos, revealed by the absence of laid embryos in the area surrounding the vulva (as these animals are paralyzed). Images were captured using a SONY CCD camera (model DMK 31AU03) with the ImagingSource software (Imaging Control).
Developmental delay
Worms expressing the gnaIs1 [Pmyo-2::yfp] and gnaIs2 [Punc-119::human Aβ; Pmyo-2::yfp] transgenes were grown at 20°C and their size was determined three days after egg-lay. Worms were imaged at 10x magnification in an Olympus BX61 fluorescence microscope equipped with a DP72 digital camera coupled to CellSens Software for image acquisition and analysis. ImageJ Software was used to quantify the length of the worms.
Quantification of α-synuclein C. elegans strains phenotypes
α-synuclein::YFP aggregates quantification
For worms expressing the rmIs126 [Punc-54::yfp] and pkIs2386 [Punc-54::α-synuclein::yfp] transgenes, the YFP aggregates were scored in the head muscle cells located within the segment encompassed by the two pharyngeal bulbs.
Dopaminergic neurodegeneration
Worms expressing the vtIs7 [Pdat-1::gfp] and baIn11 [Pdat-1::α-synuclein; Pdat-1::gfp] transgenes were scored for neurodegeneration of the six anterior dopaminergic neurons (four CEP and two ADE dopaminergic neurons) according to previously described criteria 36. Briefly, worms were scored as wild type when all six DA neurons were present and their neuronal processes were intact while worms having DA cell body loss were scored as neurodegeneration.
YFP aggregates quantification and DA neuronal integrity were assessed at 40x magnification in an Olympus BX61 fluorescence microscope equipped with a DP72 digital camera coupled to CellSens Software for image acquisition and analysis.
Paralysis
Worms expressing the baln12[Punc-54::α-synuclein::gfp] transgene (and respective controls) were grown at 20°C and paralysis scoring was initiated four days after egg-lay and determined daily, whereby paralyzed worms were removed from plates. A worm was scored as paralyzed if it did not respond to a gentle touch stimulus with a platinum wire.
Quantification of metastable protein C. elegans strains phenotypes
unc-52(ts) paralysis
Worms carrying the unc-52(e669su250) mutation were grown at 16°C and paralysis was scored at the specified days after egg-lay, whereby paralyzed worms were removed from plates. A worm was scored as paralyzed if it did not respond to a gentle touch stimulus with a platinum wire.
let-60(ts) embryonic arrest
Worms carrying the let-60(ga89) mutation were allowed to lay eggs at 16°C during 2-3 hours. Parents were removed, laid embryos counted and then allowed to grow for three days at 16°C. The embryonic arrest ratio was determined by dividing the number of developed larva by the total number of laid embryos.
HLH-30::GFP subcellular localization
Worms expressing the sqIs17 [Phlh-30::hlh-30::GFP] transgene were scored at 40x magnification at the first day of adulthood for cytoplasmic versus nuclear GFP localization in an Olympus BX61 fluorescence microscope equipped with a DP72 digital camera coupled to CellSens Software for image acquisition and analysis. For the starvation assay, animals were incubated 1 hour in M9 buffer at 20°C with gently shaking prior microscopy analysis.
SQST-1::GFP aggregates quantification
Worms expressing the bpIs151 [Psqst-1::sqst-1::GFP] transgene were allowed to lay eggs at 20°C for 2-3 hours. Parents were removed and embryos were incubated during 6 hours. Embryos with GFP aggregates were scored at 100x magnification in the same microscopy setting as above. For the starvation assay, pregnant adults were incubated in M9 buffer at 20°C with gently shaking during 24 hours. Embryos accumulated in the uterus of the starved animals were freed by dissecting the parents with a needle and scored as described above. For the RNAi assay, worms expressing the bpIs151 worms were grown on empty vector control or lgg-1 RNAi plates for 3 generations. Adults were allowed lay embryos at 20°C for 2-3 hours, further incubated during 8-10 hours until hatching and L1 larvae with more than 20 intestinal GFP aggregates were scored at 100x magnification as described above.
S. cerevisiae strains phenotypes
Yeast cells were grown at 30°C by incubation in a rotary shaker using SC medium (0.67% yeast nitrogen base, 2% glucose plus drop-out mixture and auxotrophic requirements). Experiments were performed with cell cultures growing at exponential phase (OD600= 0.5) or diauxic phase (12h after exponential phase). To image polyQ-gfp expression and aggregates wide-field fluorescence microscopy was performed on an Olympus fluorescence DP30 BW microscope (Olympus, Tokyo, Japan) equipped with a 488 nm laser excitation for GFP. Aggregation index was determined as originally described 83. Cell growth was determined in a Microplate Spectrophotometer PowerWave XS (Biotek) apparatus and values were analyzed with Gen5 1.06 software. Cells (500 μl) were grown at 30°C with continuous shaking; absorbance at 600 nm was measured each 30 min for 20 hours. Resistance to disulfide stress was tested on cells grown in SC at exponential or diauxic phase. Viability was measured by plating serial dilutions (1:10) on SC plates without or with diethyl maleate (DEM) at the indicated concentration.
SH-SY5Y cells immunofluorescence
Cells were seeded in Poly-D-lysine-coated round glass coverslips. After the treatments, they were fixed in 4% PFA for 10 minutes. Fixed cells were then blocked for 30 min in PBS (Thermo-Fisher, Massachusetts, USA) with 0.1% Triton-X and 1% bovine serum albumin. Afterwards, cells were washed three times with PBS and incubated overnight with the primary rabbit anti-Aβ42 (1:100; Lodeiro, M et al https://doi-org.proxy.kib.ki.se/10.1093/gerona/glw073). The day after, cells were washed with PBS and incubated with the secondary antibody Alexa Fluor 633 goat anti-rabbit (1:1000, Invitrogen, Corporation, Carlsbad, CA, USA) in PBS for 1h at room temperature. DAPI was used for nuclear staining. For aggregation analysis, cells were then washed thoroughly, and incubated with 0.1% (w/v) Thioflavin S in PBS for 30 min to stain for amyloid fibrils. After washing three times with PBS, cells were mounted using ProLong Gold Antifade Reagent (Thermo-Fisher, Massachusetts, USA). The primary antibody was omitted as a negative control. Confocal imaging was performed with a Zeiss LSM 510 META confocal laser scanning system. For Aβ 1 −42 quantifications, Alexa Fluor 633 fluorescence was recorded with a Zeiss LSM 510 METAL confocal laser scanning system with 20x lens (NA 0.8) and 100x (NA 1.45) lens. Quantification of fluorescence intensity in individual cells was performed with ImageJ Software. For representative figures of intracellular Aβ 1-42 aggregates, Alexa Fluor 633 fluorescence and Thioflavin-S were recorded through separate channels with 100× (NA 1.45) oil objective. Image processing was performed with the included ZEN software.
AUTHOR CONTRIBUTION
DGG, JAML and AMV designed the study, performed most experiments using C. elegans, analyzed the data and wrote the manuscript. BSN and JC performed the embryo recordings and quantified the blebbing/exploding phenotypes. FJNG and FML carried out the initial candidate RNAi screen and generated several strains. CPF and ACM performed the mammalian cell experiments. CDL generated some strains and performed paralysis and DEM toxicity experiments. CN contributed essential reagents and analysed the data. MDS and RPVM generated C. elegans strains and carried out mechanosensory assays. EFS, VG, RP and EC contributed with the yeast experiments. PA performed the embryo confocal live imaging and immunostaining. All authors edited and revised the manuscript.
COMPETING INTERESTS
Authors declare no competing interests
MOVIE LEGENDS
Movie 1
Movie recording of C. elegans wild type vs gsr-1(m−,z−) embryos. Developmental arrest of the gsr-1(m−,z−) embryo occurs at the pregastrula stage. Movie 1 corresponds to Fig. 1c.
Movie 2
Movie recording of C. elegans rmIs133 [Punc-54::Q40::yfp] embryos, that develop normally. Movie 2 corresponds to Fig. 1d.
Movie 3
Movie recording of a C. elegans rmIs133 [Punc-54::Q40::yfp]; gsr-1(m−,z−) embryo. Note the extensive cell blebbing once the embryo arrests at the pregastrula stage. Movie 3 corresponds to Fig. 1e.
Movie 4
Movie recording of C. elegans rmIs133 [Punc-54::Q40::yfp]; gsr-1(m−,z−) embryos. Note that the right embryo blebs and explodes while the left embryo explodes without undergoing blebbing. Movie 4 corresponds to Fig. 1f.
Movie 5
Movie recording of C. elegans rmls133[Punc-54::Q40::yfp]; zbls2[pie-1::lifeact-Cherry]; gsr-1(m+,z+) and gsr-1(m−,z−) embryos observed by confocal microscopy. Note that the gsr-1(m−,z−) embryos explode while the gsr-1(m+,z+) embryo completes embryogenesis and hatches. The asterisk indicates an arrested embryo that was osmotically damaged due to incomplete eggshell formation at the moment of dissection. Time is indicated in hours:minutes. Movie 5 corresponds to Fig. 1g.
Movie 6
Movie recording of C. elegans rmIs133 [Punc-54::Q40::yfp]; ltIs37 [Ppie-1::mCherry::his-58]; ojIs1 [Ppie-1::GFP::tbb-2]; ltIs24 [Ppie-1::GFP::tba-2]; gsr-1(m+,z+) and gsr-1(m−,z−) embryos observed by confocal microscopy. The left part displays GFP::tubulin whereas the middle part corresponds to mCherry::histone H2B. Abnormal accumulation of microtubules at the plasma membrane and perinuclear chromatin condensation is particularly evident in the gsr-1(m−,z−) embryo at the top. Time is indicated in hours:minutes. Movie 6 was acquired under similar conditions as the still images represented in Fig. 1h.
Movie 7
Movie recording of C. elegans wild type vs ced-10(t1875) embryos. The developmental arrest of the ced-10(t1875) embryo occurs at the premorphogenesis stage, while gsr-1(m−,z−) embryos arrest earlier, at the pregastrula stage (Movie 1). Note that the blebs formed by ced-10(t1875) embryos are not as large and prominent as those observed in gsr-1(m−,z−); rmIs133 embryos (Movies 3-4).
ACKNOWLEDGEMENTS
We thank CGC (Caenorhabiditis Genetics Center) and NBRP (National BioResource Project), Richard Morimoto, Christian Pohl, Jan Gruber, Guy Caldwell, Randy Blakely, Hong Zhang, Enriq Herrero, Anne Bertolotti and Susan Lindquist for providing strains and plasmids. Cristina Ayuso García is acknowledged for technical assistance, Leticia Lemus for help with yeast viability assays. The Spanish Ministry of Economy and Competitiveness supported EFS and VG (BFU2016-78265-P), PA (BFU2016-79313-P and MDM-2016-0687) and AMV (BFU2015-64408-P). AMV was also supported by the Instituto de Salud Carlos III (PI11/00072). All projects were cofinanced by the Fondo Social Europeo (FEDER). AMV is a member of the GENIE and EU-ROS Cost Actions of the European Union.
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