Mistranslation and ageing unmask FUS and TDP-43 toxicity in yeast models of neurodegenerative diseases

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McDonald, Nikita Chugh, Rares Sava, Martin L. Duennwald This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6448360/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Protein aggregation is indicative of the loss of proteostasis associated with neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Proteins like Fused in sarcoma (FUS) and Tar DNA-binding protein 43 (TDP-43) accumulate and aggregate in the cytosol of neurons in ALS/FTD. Yet, it remains unclear how ageing affects FUS and TDP-43 aggregation, and how these aggregates in turn influence neurodegeneration in ALS/FTD. In addition, mistranslation can reduce longevity, challenge proteostasis, and modulate protein aggregation. To investigate how ageing and mistranslation modulate FUS and TDP-43 aggregation and toxicity, we enlist tractable and reliable yeast models. We establish that respiring yeast cells are sensitized to FUS and TDP-43 compared fermenting cells, yet this increased sensitivity does not correlate with increased FUS and TDP-43 aggregation. We also demonstrate that chronological ageing and mistranslation caused by tRNA variants antagonize FUS and TDP-43 aggregation and synergize to exacerbate FUS and TDP-43 cytotoxicity. Our work provides new insights into factors that uncouple FUS and TDP-43 aggregation from toxicity and support a rather protective role for FUS and TDP-43 aggregates in promoting longevity. Protein aggregation protein misfolding molecular chaperone ageing mitochondria mistranslation ALS TDP-43 FUS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Accumulation of misfolded proteins is a hallmark of neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS) [ 1 – 3 ]. ALS manifests most commonly in sporadic cases (~ 90%, sporadic ALS, sALS), while the remaining familial cases are inherited in a mendelian fashion (familial ALS, fALS). Mutations in more than 30 different genes cause fALS, most prominently those encoding C9orf72, Superoxide Dismutase 1 (SOD1), Fused in sarcoma (FUS) and Tar DNA-binding protein 43 (TDP-43) [ 4 , 5 ]. In most ALS cases, TDP-43 [ 1 – 3 , 6 ] and, less frequently FUS [ 7 ], mis-localize from the nucleus to the cytosol where they accumulate and aggregate. The prion-like and highly intrinsically disordered domains of FUS and TDP-43 predispose these proteins to mis-localization and misfolding [ 8 – 10 ]. Also, both proteins form condensates by liquid-liquid phase separation [ 11 – 13 ], which may promote conversion of the accumulated cytosolic misfolded proteins into more stable, insoluble aggregates in the neurons of ALS patients. Of note, the role of these aggregates in ALS pathogenesis remains unclear. Key characteristics of TDP-43 and FUS, such as their misfolding, mis-localization, accumulation, and the ensuing cytotoxicity have been recapitulated in yeast models [ 8 – 10 , 14 , 15 ]. These yeast models identified genetic modulators of TDP-43 [ 16 – 24 ] and FUS [ 9 , 10 , 25 , 26 ] toxicity, leading to the discovery of modulators of ALS [ 9 , 10 , 18 , 20 , 25 ]. Yeast models have also been instrumental in deciphering the molecular and cellular mechanisms underlying TDP-43 and FUS aggregation [ 8 – 10 , 14 , 15 , 21 , 24 , 26 , 27 ]. Although the occurrence of protein aggregates in brain and spinal cord tissues are well-established neuropathological hallmarks of ALS/FTD, the causative link between these aggregates and disease progression remains tenuous. Numerous studies indicate only weak correlations between aggregation and eventual neuronal cell death [ 28 – 30 ]. Conversely, many findings even indicate potential protective functions of protein aggregates in diverse neurodegenerative diseases [ 31 , 32 ]. Accordingly, genetic modifiers of the toxicity of misfolded proteins are not necessarily modifiers of their aggregation [ 8 – 10 , 30 , 33 ]. Although TDP-43 and FUS aggregation is a clear indicator of impaired proteostasis, it remains unclear exactly how TDP-43 and FUS aggregates contribute to cellular toxicity, if at all. Moreover, the role of ageing in the formation and persistence of protein aggregates and toxicity of misfolded proteins has been notoriously difficult to ascertain. Indeed, chronological ageing hampers the ability of cells to maintain proteostasis [ 34 – 37 ]. However, the inability to form and maintain protein aggregates in aged cells is associated with reduced lifespan [ 38 ], indicating that age-dependent protein aggregates can be protective. While cultured mammalian cells are often used to model cellular senescence [ 39 ], they do not entirely recapitulate most physiological aspects of ageing. Studies of TDP-43 and FUS misfolding in the context of ageing have relied heavily on rodent models [ 40 , 41 ], which are too complex to track cellular and molecular details linking ageing to TDP-43 and FUS aggregation and toxicity. In addition to impaired proteostasis, ageing cells must contend with errors in protein biosynthesis, or mistranslation. Many have speculated [ 42 ] and shown experimentally [ 43 – 45 ] that mistranslation adversely affects longevity. While the specific mechanisms by which translation fidelity influences lifespan remain unclear, mistranslation clearly impairs proteostasis and induces protein misfolding and aggregation [ 46 – 49 ]. This plausibly contributes to the decline of proteostasis seen in ageing cells. Further, mistranslation modulates the misfolding, aggregation and toxicity of neurodegenerative disease-associated proteins, such as polyglutamine expanded huntingtin and FUS [ 50 , 51 ]. However, the degree to which ageing and mistranslation synergize to affect the misfolding of the ALS/FTD-associated proteins FUS and TDP-43 remains enigmatic. Yeast models have a rich history in the study of ageing, including the identification of key cellular factors that influence longevity, such as TOR kinase complex 1 (TOR) signaling [ 52 ], the unfolded protein response [ 35 ], reactive oxygen species[ 53 ] and metabolism [ 54 ]. Additionally, yeast models have been employed to study how translation errors influence cellular fitness and proteostasis [ 48 , 49 , 55 – 58 ]. Yet, the combination of ageing and mistranslation has not yet been studied in yeast models of misfolded proteins, including TDP-43 and FUS. To determine how ageing modulates the aggregation and toxicity of FUS and TDP-43, we establish optimized yeast models with low expression of FUS and TDP-43. These models allowed us to determine that mitochondrial respiration and β-oxidation enhances FUS and TDP-43 toxicity, but not necessarily their aggregation. Furthermore, we confirm that ageing impairs proteostasis, as evidenced by increased aggregation of a metastable reporter proteins. By contrast, ageing antagonizes FUS and TDP-43 aggregation while exacerbating the growth defect caused by FUS and TDP-43. Finally, we find that further impairing proteostasis in ageing cells by inducing translation errors synergistically prevents FUS and TDP-43 aggregation and unmask toxic phenotypes associated with FUS and TDP-43. Accordingly, our findings suggest that the loss of proteostatic capacity in ageing cells reduces aggregation of FUS and TDP-43 and thus increases their toxicity. Our findings contribute to the growing literature indicating that protein aggregates are not necessarily the major toxic species in neurodegenerative diseases, highlighting the need to investigate misfolded monomer and oligomer species as the likely culprits. Finally, our findings genetically identify translation errors, such as those caused by tRNA variants, as potential contributors or risk factors for ALS/FTD and other age-associated protein misfolding diseases. RESULTS Respiring cells are sensitive to FUS and TDP-43. Yeast models of FUS and TDP-43 aggregation and toxicity have been useful discovery tools for screening genetic interactors that antagonize toxicity [ 9 , 10 , 17 , 18 , 20 , 25 ]. However, the high degree of toxicity caused by high expression levels of FUS and TDP-43 in these models has made identifying potential enhancers of toxicity challenging. To this end, we devised models that express low levels of FUS and TDP-43 using the MET17 promoter. We compared yeast models expressing high levels of FUS and TDP-43 regulated by the common GAL1 promoter to our models expressing lower levels of FUS and TDP-43 regulated by the MET17 promoter. The MET17 promoter induced lower steady-state levels of both FUS and TDP-43 when compared to the GAL1 promoter, which corresponded with a milder growth defect (Fig. 1 A-F). Neurons are uniquely dependent on oxidative mechanisms of producing ATP for energy [ 59 , 60 ]. Yet, whether mitochondrial respiration influences the sensitivity of cells to toxicity of FUS and TDP-43 is unclear. Foregoing the GAL1 promoter in favor of the MET17 promoter enabled us to test how mitochondrial respiration influences the fitness defects caused by FUS and TDP-43. When exposed to fermentable carbon sources (glucose), yeast cells predominantly generate energy by glycolysis [ 61 , 62 ]. Instead, when forced to metabolize non-fermentable carbon sources, yeast cells generate energy by mitochondrial respiration [ 63 ]. We took advantage of the ease with which the metabolism of yeast can be manipulated to study how obligately respiring cells manage the misfolding and fitness defects associated with FUS and TDP-43. We compared cells producing energy via glycolysis (glucose) to those generating energy by oxidative phosphorylation (glycerol and potassium acetate) and β-oxidation or short (myristic acid) and long (oleic acid) chain fatty acids [ 63 ]. The fitness defects caused by FUS and FUS-YFP are exacerbated in cells grown on plates supplemented with glycerol, potassium acetate, myristic acid and oleic acid when compared to cells grown on plates supplemented with glucose (Fig. 1 G). Similarly, the fitness defect caused by TDP-43 is exacerbated by growth on glycerol, potassium acetate and myristic acid, but not oleic acid (Fig. 1 H). We next tested whether the extent of FUS and TDP-43 aggregation depends on metabolic program. We confirmed that expression of FUS-YFP and TDP43-YFP induces the formation of both FUS-YFP and TDP43-YFP foci in cells grown in glucose (Fig. 1 I-L). Aggregation of FUS-YFP was impaired in cells grown in media containing glycerol, potassium acetate and oleic acid, while myristic acid increased the number of FUS-YFP aggregates (Fig. 1 I-J). TDP43-YFP aggregation was reduced in cells grown in media containing potassium acetate, while both myristic acid and oleic acid increased the number of TDP43-YFP aggregates (Fig. 1 K-L). In sum, we generated a low expression FUS and TDP-43 yeast model. Using our novel model, we demonstrate that altering metabolic program changes the extent of aggregation and the fitness defects caused by FUS and TDP-43. Increased FUS and TDP43 toxicity in chronologically aged cells As post-mitotic cells, neurons must maintain proteostasis despite an accumulation of damage throughout their lifespan. Although protein aggregation is strongly associated with advanced age [ 34 , 38 ], our options for monitoring protein aggregation in ageing mammalian cells are limited. Model organisms provide an effective alternative to study proteostasis and disease-associated protein aggregation in ageing cells. Unlike neurons, yeast cells age through two mechanisms: chronological and replicative ageing [ 64 , 65 ]. Chronological ageing, which describes the lifespan of a yeast cell stationary phase [ 35 , 52 – 54 ], more accurately recapitulates ageing experienced by neurons than replicative ageing, which describes the number of divisions a mother cell can sustain before senescence [ 66 ]. Using our low expression FUS and TDP-43 yeast models, we investigated whether FUS and TDP-43 exacerbate the toxicity associated with chronological ageing. We first tested if ageing cells are sensitive to FUS and TDP-43 misfolding. We performed regrowth assays using cells expressing low levels of FUS or FUS-YFP that were aged for eight days. Both FUS and FUS-YFP induced a growth defect in young cells (two days of ageing) (Fig. 2 A). FUS and FUS-YFP also reduced the ability of cells to regrow after four and eight days of ageing (Fig. 2 A). We then aged cells expressing FUS or FUS-YFP for eight days in caloric restriction media (0.2% glucose), which extends lifespan in yeast [ 54 ] and monitored the ability of these cells to regrow on standard media (2% glucose). While both FUS and FUS-YFP still induced a growth defect in young cells (two days of ageing) grown in caloric restriction media, eight days of ageing did not exacerbate this growth defect (Fig. 2 B). We next performed regrowth assays with cells expressing low levels of TDP-43 and aged for a time course of eight days. TDP-43 induced a growth defect in young cells, and reduced the ability of cells to regrow after four and eight days of ageing (Fig. 2 C). The growth defect associated with TDP-43 was exacerbated after four days of ageing in caloric restriction media but was not exacerbated after eight days (Fig. 2 D). We next tested whether FUS and TDP-43 exacerbated cell death associated with ageing. Using propidium iodide staining as a proxy for cell death, we measured the percentage of dead cells in control cells and cells expressing FUS or TDP-43 after two, four and eight days of ageing. Expression of FUS did not change the percentage of dead cells when compared to control cells at any point of ageing (Fig. 2 E). In contrast, TDP-43 mildly reduced the percentage of dead cells when compared to control cells after eight days of ageing (Fig. 2 F). Ageing cells in caloric restriction media reduced the percentage of dead cells after eight days of ageing, regardless of the expression of FUS or TDP-43 (Fig. 2 E-F). We performed western blots to test whether ageing changes steady state levels of FUS and TDP-43. Steady state levels of FUS and TDP-43 did not change when cells were aged (Fig. 2 G-H). We then turned our attention to how FUS and TDP-43 influence expression of molecular chaperones in ageing cells. We found that expression of FUS and TDP-43 reduced steady state levels of Hsp104, Hsp42 and Hsp26 when compared to control cells after two days of ageing (Fig. 2 G-K). Cells expressing FUS maintained lower levels of Hsp104 and Hsp42 after four days of ageing, while Hsp26 levels recovered back to control levels (Fig. 2 G-K). In contrast, cells expressing TDP-43 maintained lower levels of Hsp42 and Hsp26, while Hsp104 levels recovered back to control levels (Fig. 2 G-K). In sum, both FUS and TDP-43 impair the fitness of aged cells, although neither FUS nor TDP-43 exacerbate cell death caused by ageing. Furthermore, both FUS and TDP-43 reduce steady state levels of Hsp104, Hsp42 and Hsp26, possibly further impairing proteostasis in ageing cells. Ageing reduces FUS and TDP-43 inclusions. Neurodegenerative phenotypes have long been associated with aggregation of misfolded proteins, like FUS and TDP-43, in ageing neurons. We sought to unravel the relationship between chronological ageing and aggregation of FUS and TDP-43. To this end, we monitored aggregation of FUS-YFP and TDP43-YFP during the aging process. We aged cells expressing FUS-YFP in standard media (2% glucose) and caloric restriction media (0.2% glucose) for a time course of four days. Both the percentage of cells with FUS-YFP foci and the number of FUS-YFP foci per cell were reduced after four days of ageing (Fig. 3 A-C). Caloric restriction also reduced the percentage of cells with FUS-YFP foci but masked the reduction in FUS-YFP foci caused by ageing (Fig. 3 A-C). Although low levels of FUS does not induce cell death, we hypothesized that the ability of cells to maintain FUS aggregates may influence the population of cells that die. To test this, we next stained cells expressing FUS-YFP with propidium iodide to assess the occurrence of FUS-YFP inclusions in dying cells. Across all conditions, cells stained with propidium iodide less frequently contained FUS-YFP foci when compared to their unstained counterparts (Fig. 3 A-B). We next aged cells expressing TDP43-YFP for four days. Similarly to FUS-YFP, both the percentage of cells with TDP43-YFP foci and the number of TDP43-YFP foci per cell were reduced after four days of ageing (Fig. 3 D-F). In contrast to FUS-YFP, the percentage of cells with TDP43-YFP foci was not reduced by caloric restriction (Fig. 3 D-F). In addition, when monitoring cells stained with propidium iodide, we find that cells stained with propidium iodide less frequently contained TDP43-YFP foci when compared to their unstained counterparts (Fig. 3 D-E). To confirm that ageing impairs proteostasis, we aged cells expressing metastable Firefly Luciferase-GFP (FFL-GFP) as proteostasis reporter that is not directly linked to neurodegenerative diseases. The percentage of cells with FFL-GFP foci and the number of FFL-GFP foci per cell increased after four days of ageing (Fig. S1 A-C). To assess whether caloric restriction influences proteostasis decline in ageing cells, we aged cells expressing FFL-GFP in caloric restriction media. Caloric restriction did not change the percentage of cells with FFL-GFP foci, nor the number of FFL-GFP foci per cell (Fig. S1 A-C). As dead cells lose the ability to maintain FUS and TDP-43 inclusions, we sought to assess whether they also lose the ability to maintain FFL-GFP inclusions. Similar to FUS-YFP and TDP43-YFP, cells stained with propidium iodide showed a drastic reduction in the percentage of cells with FFL-GFP foci (Fig. S1 A-B). In sum, our findings indicate a reduction in both FUS and TDP-43 aggregation in ageing cells. In contrast, FFL aggregation increases in ageing cells, both showing specificity and confirming impaired proteostasis in ageing cells. Finally, our findings reveal a negative correlation between FUS, TDP-43 and FFL aggregation and cell death. FUS and TDP-43 disrupt molecular chaperone solubility. Molecular chaperones are major modulators of protein folding and aggregation [ 67 ]. In particular, the AAA-ATPase disaggregase Hsp104 and the holdases Hsp42 and Hsp26 cooperate to regulate protein aggregation in yeast cells. Importantly, loss of Hsp104, Hsp42 and Hsp26 impairs fitness of ageing cells [ 64 ]. We explored whether FUS and TDP-43 dysregulate the function of Hsp104, Hsp42 and Hsp26 in ageing cells, leading to loss of aggregation. First, we monitored the solubility of FUS and TDP-43 to confirm loss of FUS and TDP-43 aggregation in ageing cells. We performed sedimentation assays using ageing cells expressing either FUS or TDP-43 to separate proteins into soluble (s) and insoluble (p) fractions. Both FUS and TDP-43 were less present in the insoluble fraction, and thus more soluble, after four days of ageing (Fig. 4 A-C). We next turned our attention to the solubility of Hsp104, Hsp42 and Hsp26 in ageing cells expressing FUS and TDP-43. Expression of FUS, but not TDP-43, reduced the solubility of Hsp104 when compared to control cells (Fig. 4 D-G). In both control cells and, to a lesser extent, cells expressing FUS, Hsp104 became more soluble after four days of ageing (Fig. 4 D-E, 4 G). However, in cells expressing TDP-43, Hsp104 became less soluble after four days of ageing (Fig. 4 F-G). Both FUS and TDP-43 reduced the solubility of Hsp42 when compared to control cells (Fig. 4 D-F, 4 H). While Hsp42 became less soluble after four days of ageing in control cells, Hsp42 became more soluble after four days of ageing in cells expressing FUS, but not TDP-43 (Fig. 4 D-F, 4 H). In similar fashion to Hsp42, both FUS and TDP-43 reduced the solubility of Hsp26 when compared to control cells (Fig. 4 D-F, 4 I). In both control cells and, to a lesser extent, cells expressing FUS, Hsp26 became less soluble after four days of ageing (Fig. 4 D-E, 4 I). In contrast, Hsp26 solubility did not change in cells expressing TDP-43 after four days of ageing (Fig. 4 F, 4 I). We next monitored FUS-mCherry and TDP43-mCherry aggregation in cells deleted for Hsp104 ( Δhsp104 ) or Hsp42 and Hsp26 ( Δhsp42Δhsp26 ). Loss of Hsp104 reduced the percentage of cells with FUS-mCherry and TDP43-mCherry foci and the number of FUS-mCherry and TDP43-mCherry foci per cell (Fig. S2A-C). However, loss of Hsp42 and Hsp26 only reduced the percentage of cells with TDP43-mCherry foci and the number of TDP43-mCherry foci per cell (Fig. S2A-C). We also performed growth assays to determine whether loss of FUS and TDP-43 aggregation affects growth. Loss of Hsp104 did not change the growth of cells expressing FUS or TDP-43 (Fig. S2D-E). However, loss of Hsp42 and Hsp26 exacerbated the growth defect caused by FUS, but rescued the growth defect caused by TDP-43. As both FUS and TDP-43 modulate the solubility of molecular chaperones, we tested whether FUS and TDP-43 impair proteostasis in ageing cells. We expressed a metastable FFL-mCherry reporter in ageing cells co-expressing FUS and TDP-43. FUS and TDP-43 do not increase the percentage of cells with FFL-mCherry foci, nor the number of FFL-mCherry foci per cell in young cells (Fig. 4 J-L). However, after four days of ageing, FUS increased the number of FFL-mCherry foci per cell when compared to control cells (Fig. 4 J, 4 L). In stark contrast, TDP-43 decreased both the percentage of cells with FFL-mCherry foci and the number of FFL-mCherry foci per cell when compared to control cells (Fig. 4 J-L). In sum, both FUS and TDP-43 reduce the solubility of the molecular chaperones Hsp104, Hsp42 and Hsp26 in ageing cells. Furthermore, loss of Hsp104, Hsp42 and Hsp26 function impairs aggregation of FUS and TDP-43, indicating a potential mechanism underlying the reduced aggregation of FUS and TDP-43 in ageing cells. Finally, although both FUS and TDP-43 reduce solubility of molecular chaperones, they have different effects on protein folding, as measured by FFL-mCherry aggregation. Mistranslation unmasks toxicity of FUS and TDP-43 in ageing cells. The ability of organisms to maintain translation fidelity is strongly linked to increased longevity [ 43 – 45 ]. Inducing translation errors (mistranslation) is detrimental to proteostasis [ 46 , 49 , 56 – 58 ], and can even modulate aggregation of endogenous proteins [ 47 ], metastable proteins [ 48 ], and proteins associated with neurodegenerative diseases like polyglutamine expanded huntingtin protein [ 50 ] and FUS [ 51 ]. However, how mistranslation impacts proteostasis in ageing cells is poorly understood. We sought to investigate whether mistranslating tRNA variants affect chronological lifespan of cells. To induce mistranslation, we expressed serine tRNA variants with anticodon mutations that induce proline to serine misincorporation at rates of approximately 0.5% (P > S weak) or 5% (P > S strong), or arginine to serine misincorporation at a rate of approximately 3.5% (R > S) [ 57 ]. We performed growth assays using cells expressing mistranslating tRNA variants and aged over eight days in either standard media (2% glucose) or caloric restriction media (0.2% glucose). P > S strong and R > S both impair the ability of cells to regrow after eight days of ageing in standard media, but not caloric restriction media (Fig. 5 A-B). We stained mistranslating cells with propidium iodide to measure the percentage of dead cells. While mistranslating tRNA variants did not exacerbate cell death in cells aged in standard media, P > S strong and R > S partially antagonized the protective effects of caloric restriction (Fig. 5 C-D). We explored whether mistranslating tRNA variants augment the toxicity associated with FUS and TDP-43 in ageing cells. We aged cells co-expressing mistranslating tRNA variants and either FUS or TDP-43 for eight days. P > S strong and R > S synergistically impaired the ability of cells expressing FUS and TDP-43 to regrow after eight days of ageing (Fig. 5 E-F). P > S strong and R > S also induces synthetic cell death in cells expressing FUS and TDP-43 after four and eight days of ageing (Fig. 5 G-H). In sum, mistranslating tRNA variants impair the fitness of ageing cells, but they do not exacerbate cell death when expressed in isolation in ageing cells. However, when expressed in addition to FUS and TDP-43, mistranslation synergistically impairs fitness of ageing cells by inducing synthetic cell death. Mistranslation reduces FUS and TDP-43 inclusions in ageing cells. Thus far, we have demonstrated an inverse correlation between ageing and inclusion formation of FUS and TDP-43, indicating a potential protective function of FUS and TDP-43 inclusions. We speculated that the synergistic toxicity of mistranslating tRNA variants and FUS and TDP-43 in ageing cells is due to an inability of cells to form protective inclusion of FUS and TDP-43. To test this hypothesis, we monitored FUS-YFP and TDP43-YFP aggregation in ageing cells expressing mistranslating tRNA variants. P > S weak, P > S strong and R > S all reduced the percentage of cells containing FUS-YFP foci, while P > S strong and R > S reduced the number of FUS-YFP foci per cell (Fig. 6 A-C). After ageing cells for four days, P > S weak, P > S strong and R > S led to a greater decrease in the percentage of cells with FUS-YFP foci and the number of FUS-YFP foci per cell when compared to cells expressing wt tSer (Fig. 6 A-C). While mistranslating tRNA variants had no effect on the percentage of cells with TDP43-YFP foci, P > S strong and R > S both reduced the number of TDP43-YFP foci per cell (Fig. 6 D-F). Although four days of ageing reduced both the percentage of cells with TDP43-YFP foci and the number of TDP43-YFP foci per cell regardless of the tRNA expressed, mistranslating tRNA variants did not change the percentage of cells with TDP43-YFP foci and the number of TDP43-YFP foci per cell after four days of ageing (Fig. 6 D-F). We used the metastable FFL-GFP reporter to test whether mistranslating tRNA variants reduces aggregation of FUS and TDP-43 by impairing proteostasis. P > S strong and R > S both increased the percentage of cells containing FFL-GFP foci, but not the number of FFL-GFP foci per cell in young cells (Fig. S3A-C). After ageing cells for four days, P > S strong and R > S both led to a greater increase in the number of FFL-GFP foci per cell, but not the percentage of cells containing FFL-GFP foci, when compared to cells expressing wt tSer. In sum, our findings suggest that mistranslating tRNA variants reduce aggregation of FUS and TDP-43 in both young and aged cells, supporting our hypothesis that FUS and TDP-43 inclusions are protective. In contrast, mistranslating tRNA variants increase aggregation of FFL-GFP in young and aged cells, confirming that mistranslation impairs proteostasis and indicating that impaired proteostasis inhibits aggregation of FUS and TDP-43. DISCUSSION Like many other neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS) are associated with the accumulation of aggregated proteins, such as Fused in sarcoma (FUS) [ 7 ] and Tar DNA-binding protein 42 (TDP-43) [ 1 , 2 , 6 ]. Accordingly, protein aggregates had been proposed to act as key drivers of neurodegeneration and age-related cell death, generally [ 1 , 3 , 68 , 69 ]. However, many detailed studies challenge the notion that protein aggregation causes toxicity in models of ageing and neurodegeneration [ 28 – 31 ]. Additionally, fidelity of protein biosynthesis is clearly a modifier of ageing [ 43 – 45 ] and protein aggregation [ 47 – 49 ], including aggregation of proteins associated with neurodegenerative diseases [ 50 , 51 ]. Yeast has been an effective model to study chronological ageing [ 35 , 52 , 54 , 64 ], translation errors [ 48 , 49 , 56 , 70 ], and aggregation of neurodegenerative disease-associated proteins [ 8 – 10 , 14 , 16 , 33 ]. Here, we explore the combined influence of ageing and translation errors on aggregation of FUS and TDP-43 and their toxicity, which closely reflects the accumulation of different proteostasis stressors experienced over the lifespan of neurons in ALS. Previously described FUS and TDP-43 yeast models expressed very high levels of FUS and TDP-43 [ 8 – 10 , 14 ], which have delivered important insights into aggregation and cytotoxicity of FUS and TDP-43. However, owing to their acute cytotoxicity, these models are not optimal for investigating metabolism and chronological ageing as modifiers of FUS and TDP-43 aggregation and their toxicity. We overcome these limitations by devising yeast models that express low levels of FUS and TDP-43, resulting only mild growth defects. Furthermore, our optimized yeast models facilitate studies in media containing non-fermentable carbon sources, driving yeast cells to energy production via mitochondrial respiration [ 63 ], which mimics the metabolic activity in neurons [ 59 , 60 ]. We show that cells producing energy by oxidative phosphorylation or β-oxidation are sensitized to expression of both FUS and TDP-43 compared to fermenting yeast. Notably, respiring cells frequently show a reduction in FUS and TDP-43 aggregates, suggesting an uncoupling of FUS and TDP-43 aggregation from their cytotoxicity. Therefore, we speculate that aggregation is not necessarily the key mechanism by which FUS and TDP-43 induce toxicity in respiring cells. Our study further explores how chronological ageing alters the accumulation of FUS and TDP-43 aggregates and their toxicity. When cells are aged chronologically, we find that FUS and TDP-43 do not induce cell death, but instead prevent aged cells from re-entering the cell cycle. Further, we reveal a negative correlation between ageing and FUS and TDP-43 aggregation. Notably, protein aggregation was not universally inhibited by ageing, as ageing increased the aggregation of a commonly used metastable reporter protein, Firefly Luciferase-GFP (FFL-GFP). These findings may reveal a specific impact of ageing on the RNA granule condensates that TDP-43 and FUS associate with[ 9 , 15 ] compared to the spatial quality control compartments that aggregated FFL is sequestered to [ 71 ]. Moreover, cells that die during the ageing process consistently retain fewer FUS and TDP-43 aggregates compared to their surviving counterparts. We propose that FUS and TDP-43 aggregates perform protective functions, and that their presence may be predictive of cell survival. Accordingly, we postulate that ageing cells are particularly sensitive to the misfolding of FUS and TDP-43, plausibly due to impaired proteostasis[ 34 , 36 , 72 ] an inability to form or maintain protective aggregates [ 38 ]. We also assessed whether FUS and TDP-43 interact with proteostasis networks to impair their aggregation in ageing cells. Indeed, FUS and TDP-43 reduce the steady-state levels and the solubility of molecular chaperones, thereby likely reducing their protective functions in proteostasis and compromising longevity [ 37 , 64 , 72 – 76 ]. We furthermore investigate whether mistranslating tRNA variants exacerbate ageing phenotypes in our FUS and TDP-43 yeast models. Our findings indicate that, while mistranslating tRNA variants do not induce cell death, they prevent aged cells form re-entering the cell cycle. However, mistranslating tRNA variants partially reduce the protective effect of caloric restriction on age-related cell death. Remarkably, although neither mistranslating tRNA variants, nor FUS and TDP-43, exacerbate age-related cell death, the combination of mistranslation and FUS or TDP-43 induces synthetic toxicity in ageing cells. Notably, the stress caused by mistranslating tRNA variants closely mimics that of ageing, impairing proteostasis and increasing aggregation of FFL-GFP, while antagonizing the aggregation of FUS and TDP-43. We deduce that ageing, mistranslation and expression of FUS or TDP-43 synergistically impair proteostasis, preventing formation of protective FUS and TDP-43 aggregates and foiling cellular survival. These findings support our hypothesis that the inability to maintain FUS and TDP-43 aggregates in ageing cells promotes their toxicity. CONCLUSION Most ALS patients accumulate aggregated proteins, like TDP-43 and FUS, in their neurons. The mechanism by which ageing modulates this aggregation, and whether these aggregates cause disease has remained elusive. Here, we find that ageing, paradoxically but specifically, impairs aggregation of FUS and TDP-43, plausibly due to impaired proteostasis. We also highlight mistranslation as a modulator of FUS and TDP-43 toxicity in ageing cells. Based on our findings, we propose a model, whereby mistranslation and possibly other challenges to proteostasis, combined with ageing impair aggregation, thus unleashing the cytotoxicity associated with smaller, more soluble misfolded FUS and TDP-43 conformers. Our research underscores the need to determine the molecular and cellular mechanisms underlying protective protein aggregation and explore if this model also to other misfolded proteins and neurodegenerative diseases. MATERIALS AND METHODS Plasmids All plasmids used in this study are described in Supplementary Table 1. The plasmids encoding TDP-43, FUS and FUS-YFP under control of the MET17 promoter were generated using cut-paste cloning by Analisa Echeverria (this study). The plasmid encoding TDP43-YFP was generated using Gateway cloning. The plasmid encoding FUS under control of the GAL1 promoter was a kind gift from Dr. Aaron Gitler [ 9 ]. The plasmid encoding TDP-43 under control of the GAL1 promoter was generated by Gateway cloning. The p426MET17 FFL D50N,G119N -GFP plasmid was a kind gift from Dr. John Glover [ 77 ]. The plasmids encoding TDP-43, FUS and FFL D50N,G119N -mCherry were all generated using Gateway cloning using a pAG416GPD ccdB mCherry destination vector, which was designed using cut-paste cloning. Plasmids encoding tRNA variants were a kind gift from Dr. Chris Brandl (pSUP17-wt, pSUP17-P > S weak , pSUP17-P > S strong , pSUP17-R > S) [ 57 ]. Yeast strains, media and culturing conditions All experiments were performed using derivatives of the W303 yeast strain ( leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 ). Strains deleted for HSP104, or HSP42 and HSP26 were described previously [ 78 ]. Yeast cells were transformed with desired plasmids encoding either FUS, TDP-43 or FFL D50N,G119N using the standard LiAc/PEG protocol [ 79 ]. Cells were cultured in and plated on agar plates containing synthetic defined (SD) media supplemented with 2% glucose, 6.7 g/mL yeast nitrogenous bases with ammonium sulfate and amino acids (40 mg/mL L-lysine, 20 mg/mL L-arginine, 10 mg/mL L-threonine, 60 mg/mL L-phenylalanine, 20 mg/mL L-isoleucine, 10 mg/mL L-methionine, 20 mg/mL adenine hemisulfate). Media was supplemented with additional amino acids for selection where necessary (20 mg/mL L-histidine, 60 mg/mL L-leucine, 20 mg/mL uracil, 80 mg/mL L-tryptophan). L-methionine was omitted from the media when necessary for induction of the MET17 promoter. For ageing experiments, cells were grown to saturation in a 3mL liquid culture for 24 hours before cells were pelleted and resuspended in media lacking methionine. Cells were then allowed to age chronologically for eight days. At the indicated time points, aliquots of the culture were taken to perform experiments. For caloric restriction, cells were grown to saturation in a 3 mL liquid culture for 24 hours before the cells were pelleted and resuspended in media lacking methionine and containing only 0.2% glucose. Growth assay To assess growth defects, we performed growth assays as described by Petropavlovskiy et al. [ 80 ]. Briefly, cells were diluted to OD600 = 1 and 5-fold serial dilutions were performed of each sample in a 96-well plate. Using a 48-pronged frogger, single spots of each sample were plated onto SD agar plates containing 2% glucose. When stated, cells were plated on agar plates that were modified to contain different carbon sources (2% galactose, 2% glycerol, 2% potassium acetate (KoAc), 0.1% myristic acid solubilized in 0.05% Tween20, or 0.1% oleic acid solubilized in 0.05% Tween20) instead of 2% glucose. Plates were incubated at 30°C for 24–72 hours before plates were photographed and analyzed using ImageJ. The densitometry of the spots at the same dilution factor were later compared. Experiments were repeated at least three times with independent transformants. Propidium iodide staining and flow cytometry To assess cell death, we performed propidium iodide staining as described by Chadwick et al. [ 64 ]. Briefly, 100 µL of cell culture was aliquoted into a labelled tube, pelleted and resuspended in 100 µL of 5 µg/mL propidium iodide in PBS. After 10 minutes, the cells were pelleted and resuspended in PBS. The percentage of propidium iodide-stained cells was determined by flow cytometry using the BD Bioscience FACS Celesta flow cytometer. A 561 nm laser was used to identify cells stained with propidium iodide. A sample of cells that were boiled for five minutes was used to generate a gate corresponding to propidium iodide positive staining. At least three independent experiments were performed for each sample, with a minimum of 150,000 cells being analyzed. Fluorescence microscopy To measure the formation of protein inclusions, we performed fluorescence microscopy using cells expressing either FFL-GFP or FUS-YFP. Briefly, 100 µL of cell culture was aliquoted into a labelled tube and stained with propidium iodide as previously described. Cells were then imaged using the BioTek Cytation 5 Cell Imaging Multi-mode Reader at 20x magnification. Images were taken using a GFP filter to visualize and assess inclusion formation, and a TexasRed filter to visualize and assess cells stained with propidium iodide. Experiments were repeated at least three times with independent transformants and at least 400 cells were imaged and later analyzed. Downstream analysis of fluorescence microscopy images was performed in R using the EBImage package in R [ 81 ]. Protein extraction and Western Blot To measure steady state levels of proteins, protein extraction was performed using a modified alkaline lysis protocol [ 82 ]. Briefly, 0.5 mL of OD600 = 2 cells were harvested and resuspended in 50 µL alkaline lysis buffer (0.1M NaOH, 2% SDS, 50mM EDTA) and boiled for five minutes. The lysate was then cleared at 21k xg for 10 minutes, transferred to a fresh tube and diluted in loading buffer and boiled again for five minutes. Protein lysates were then resolved by SDS-PAGE and transferred to PVDF membrane followed by immunoblotting with anti-FUS (Bethyl, A300-302A, 1:500), anti-TDP-43 (Novus, H00023435-M01, 1:500), anti-Pgk1 (Origene, AP21371AF-N, 1:5000), anti-Hsp104, anti-Hsp42 and anti-Hsp26 (all kind gifts from Dr. Johannes Buchner). Secondary antibodies used were either conjugated with Alexa680 (Thermofisher, 1:5000) or HRP (Thermofisher, 1:5000). Blots were imaged and the densitometry of bands was assessed and compared in relation to the Pgk1 loading control. Experiments were repeated at least three times with independent transformants. Sedimentation assay To assess solubility of proteins, a mild mechanical lysis was used to extract proteins. Briefly, 3 mL of OD600 = 2 cells were harvested and resuspended in 100 µL mild lysis buffer (100 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM DTT, 5 mM EDTA, 50 µM NEM and 10 µM PMSF). Cell suspension was then transferred to an Eppendorf tube containing 100 µL acid-washed glass beads (Sigma, G8772) and then disrupted for five 1-minute cycles interspersed by 1-minute incubations on ice. The lysate was then cleared at 200 xg for 2 minutes before being transferred to a new tube. An aliquot of the total lysate was taken before the remaining lysate was centrifuged at 500 xg for 15 minutes. The supernatant was aliquoted into a new tube and the pellet was then resuspended in mild lysis buffer. All protein fractions were diluted with an equal volume of SUMEB (1% SDS, 8 M Urea, 10 mM MOPS, 10 mM EDTA). Equal volumes of each protein fraction were then resolved by SDS-PAGE followed by immunoblotting as described above. Statistical analysis All graphical representations of data and statistical analysis were performed using GraphPad Prism. Data were compared using either one or two-way ANOVA followed by Fisher’s LSD. All relevant p-values are stated. Declarations COMPETING INTERESTS We have no competing interests to declare. Author Contribution Donovan W. McDonald: Investigation, Conceptualization, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. Nikita Chugh: Investigation, Formal analysis. Rares Sava: Investigation, Formal analysis. Martin L. Duennwald: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing—review & editing. Acknowledgement We would like to thank Analisa Echeverria (Boston Biomedical) for generating the MET17 driven TDP-43, FUS and FUS-YFP plasmids. We would also like to thank Drs. Aaron D. Gitler (Stanford University), John R. Glover (University of Toronto) and Christopher J. Brandl (University of Western Ontario, Emeritus) for providing plasmids used in this study. We would also like to thank Drs. Patrick Lajoie (University of Western Ontario) and Emily M. Sontag (Marquette University) for their critical editing of this manuscript. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The R script used to analyze fluorescence microscopy is available at https://doi.org/10.6084/m9.figshare.28688966. References Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al. Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. 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McDonald","email":"","orcid":"","institution":"The University of Western Ontario","correspondingAuthor":false,"prefix":"","firstName":"Donovan","middleName":"W.","lastName":"McDonald","suffix":""},{"id":454876312,"identity":"9466beb2-20bd-464e-9f95-529349c93133","order_by":1,"name":"Nikita Chugh","email":"","orcid":"","institution":"The University of Western Ontario","correspondingAuthor":false,"prefix":"","firstName":"Nikita","middleName":"","lastName":"Chugh","suffix":""},{"id":454876313,"identity":"afa1a6bd-ed44-40a9-9ca1-47c54d979359","order_by":2,"name":"Rares Sava","email":"","orcid":"","institution":"The University of Western Ontario","correspondingAuthor":false,"prefix":"","firstName":"Rares","middleName":"","lastName":"Sava","suffix":""},{"id":454876314,"identity":"bd7ef2ea-c527-4b2c-b78f-4a4d4db4209b","order_by":3,"name":"Martin L. Duennwald","email":"data:image/png;base64,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","orcid":"","institution":"The University of Western Ontario","correspondingAuthor":true,"prefix":"","firstName":"Martin","middleName":"L.","lastName":"Duennwald","suffix":""}],"badges":[],"createdAt":"2025-04-14 17:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6448360/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6448360/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82645933,"identity":"37592704-f2ab-47b5-bc6e-6eb4b670e85e","added_by":"auto","created_at":"2025-05-13 16:05:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2292599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial respiration exacerbates fitness defect caused by FUS and TDP-43\u003c/strong\u003e. A. Growth assays of cells expressing FUS under control of the MET17 or GAL1 promoters. B. Western blot and C. quantification of lysates from SH-SY5Y cell line or yeast cells expressing vector control or FUS under control of the MET17 or GAL1 promoter. D. Growth assays of cells expressing TDP-43 under control of the MET17 or GAL1 promoters. E. Western blot and F. quantification of lysates from SH-SY5Y cell line or yeast cells expressing vector control or TDP-43 under control of the MET17 or GAL1 promoter. G. Growth assays of cells expressing vector control, FUS-YFP or FUS on plates containing non-fermentable carbon sources. H. Growth assays of cells expressing either vector control or TDP-43 on plates containing non-fermentable carbon sources. I. Fluorescence microscopy of cells expressing FUS-YFP grow in media containing non-fermentable carbon sources. J. Quantification of the number of FUS-YFP foci per cell. K. Fluorescence microscopy of cells expressing TDP43-YFP grown in media containing non-fermentable carbon sources. L. Quantification of the number of TDP43-YFP foci per cell. The means (+/- SD) are represented graphically for three independent experiments. For microscopy, at least 500 cells were analyzed for each condition. # symbols indicate synergistic interactions when compared to the same sample grown in glucose media (\u003cem\u003e# p\u0026lt;0.05, ## p\u0026lt;0.01, ### p\u0026lt;0.001, #### p\u0026lt;0.0001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/acd78cbbae1d398a017ef2f7.png"},{"id":82645946,"identity":"34fb061d-b69b-4027-9ed2-6430ea135acf","added_by":"auto","created_at":"2025-05-13 16:05:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2411549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChronological ageing exacerbates growth defects associated with FUS and TDP-43\u003c/strong\u003e. A-B. Growth assays of cells expressing either vector control, FUS-YFP or FUS after two, four or eight days of ageing in media containing A. 2% glucose or B. 0.2% glucose. C-D. Growth assays of cells expressing either vector control or TDP-43 in media containing C. 2% glucose or D. 0.2% glucose. E. Flow cytometry measuring the percentage of cells stained for propidium iodide in the population of cells expressing either vector control or FUS aged for two, four or eight days in media containing either 2% glucose or 0.2% glucose. F. Flow cytometry measuring the percentage of cells stained for propidium iodide in the population of cells expressing either vector control or TDP-43 aged for two, four or eight days in media containing either 2% glucose or 0.2% glucose. G. Western blot of lysates from control cells or cells expressing FUS or TDP-43 after two and four days of chronological ageing. H-K. Quantification of H. FUS \u0026amp; TDP-43, I. Hsp104, J. Hsp42 and K. Hsp26 steady state levels. The means (+/- SD) are represented graphically for three independent experiments. For flow cytometry, 200,000 cells were analyzed for each condition. # symbols indicate synergistic interactions when compared to the same sample at Day 2 (\u003cem\u003e# p\u0026lt;0.05, ## p\u0026lt;0.01, ### p\u0026lt;0.001, #### p\u0026lt;0.0001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/a3fe9058e8cb55173807a8b6.png"},{"id":82645927,"identity":"30575473-376d-4134-8fef-f96124490fae","added_by":"auto","created_at":"2025-05-13 16:05:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1914896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-correlation of ageing and cell death with aggregation of FUS and TDP-43\u003c/strong\u003e. A. Fluorescence microscopy of cells expressing FUS-YFP aged in media containing 2% or 0.2% glucose for two or four days and subsequently stained with propidium iodide. B. Quantification of the percentage of cells containing FUS-YFP foci. C. Quantification of the number of FUS-YFP foci per cell. D. Fluorescence microscopy of cells expressing TDP43-YFP aged in media containing 2% or 0.2% glucose for two or four days and subsequently stained with propidium iodide. E. Quantification of the percentage of cells containing FUS-YFP foci. F. Quantification of the number of FUS-YFP foci per cell. The means (+/- SD) are represented graphically for three independent experiments. At least 500 cells were analyzed for each condition.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/4921cec807b85472b28057a6.png"},{"id":82645930,"identity":"dc6c9280-84c5-4079-aa69-8bbcfdb5006b","added_by":"auto","created_at":"2025-05-13 16:05:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1988358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFUS and TDP-43 impair protein quality control in ageing cells\u003c/strong\u003e. A. Sedimentation assay of FUS and TDP-43 in ageing cells. B-C. Quantification of the ratio of B. FUS and C. TDP-43 in supernatant compared to the pellet. D-F. Sedimentation assay of lysates extracted from D. control cells or cells expressing E. FUS or F. TDP-43. G-I. Quantification of the ratio of G. Hsp104, H. Hsp42 and I. Hsp26 in the supernatant compared to the pellet. J. Fluorescence microscopy of cells expressing FFL-mCherry and either vector control, FUS or TDP-43 after two and four days of ageing. K. Quantification of the percentage of cells containing FFL-mCherry foci. L. Quantification of the number of FFL-mCherry foci per cell. The means (+/- SD) are represented graphically for three independent experiments. For microscopy, at least 500 cells were analyzed for each condition. * symbols indicate significant differences at Day 4 when compared to Day 2 (\u003cem\u003e* p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/29a72db92b2171309d4632d9.png"},{"id":82645938,"identity":"1b37799e-f402-4e84-ac0d-499004521987","added_by":"auto","created_at":"2025-05-13 16:05:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2497265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMistranslation unmasks toxicity of FUS and TDP-43 in ageing cells. \u003c/strong\u003eA-B. Growth assays of cells expressing either vector control, wt tSer or the mistranslating tSer variants P\u0026gt;S weak, P\u0026gt;S strong or R\u0026gt;S after two, four or eight days of ageing in media containing A. 2% glucose or B. 0.2% glucose. C-D. Flow cytometry measuring the percentage of cells stained with propidium iodide in the population of cells expressing either vector control, wt tSer or mistranslating tSer variants after two, four or eight days of ageing in media containing C. 2% glucose or D. 0.2% glucose. E-F. Growth assays of cells expressing either vector control, wt tSer, or mistranslating tSer variants and either E. FUS or F. TDP-43 after two, four or eight days of ageing. G-H. Flow cytometry measuring the percentage of cells stained with propidium iodide in the population of cells expressing either vector control, wt tSer or mistranslating tSer variants and either E. FUS or F. TDP-43 after two, four or eight days of ageing. The means (+/- SD) are represented graphically for three independent experiments. For flow cytometry, at least 150,000 cells were analyzed for each condition.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/cc96132fd4fbf60af49f8784.png"},{"id":82645932,"identity":"bc7b38ea-0453-4bb0-b935-c3edf67599dc","added_by":"auto","created_at":"2025-05-13 16:05:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1586835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranslation errors reduce inclusion formation of FUS and TDP-43 in ageing cells. \u003c/strong\u003eA. Fluorescence microscopy of cells expressing FUS-YFP and either wt tSer or the mistranslating tSer variants P\u0026gt;S weak, P\u0026gt;S strong or R\u0026gt;S aged for two or four days. B. Quantification of the percentage of cells with FUS-YFP foci. C. Quantification of the number of FUS-YFP foci per cell. D. Fluorescence microscopy of cells expressing TDP43-YFP and either wt tSer or the mistranslating tSer variants P\u0026gt;S weak, P\u0026gt;S strong or R\u0026gt;S aged for two or four days. E. Quantification of the percentage of cells with FUS-YFP foci. F. Quantification of the number of FUS-YFP foci per cell. The means (+/- SD) are represented graphically for three independent experiments. At least 500 cells were analyzed for each condition. (\u003cem\u003e* p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/1dd6f1359abc83494c94bcef.png"},{"id":82648228,"identity":"8a5be645-dc58-47ed-b2cb-d0506a6c5931","added_by":"auto","created_at":"2025-05-13 16:37:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13518659,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/1e60f950-f5f3-454c-91ec-63d8fd708ba7.pdf"},{"id":82645926,"identity":"50e4f267-088e-4c04-913b-53096c8e8537","added_by":"auto","created_at":"2025-05-13 16:05:10","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10651,"visible":true,"origin":"","legend":"","description":"","filename":"BMCBiolSuppTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/ab946375b7e4d1236b58143c.xlsx"},{"id":82645928,"identity":"20cbfca9-7448-47d7-b386-c6e100bc79bd","added_by":"auto","created_at":"2025-05-13 16:05:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":505430,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6448360/v1/b8d2c1ad04627085e0426c3b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mistranslation and ageing unmask FUS and TDP-43 toxicity in yeast models of neurodegenerative diseases","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAccumulation of misfolded proteins is a hallmark of neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS) [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. ALS manifests most commonly in sporadic cases (~\u0026thinsp;90%, sporadic ALS, sALS), while the remaining familial cases are inherited in a mendelian fashion (familial ALS, fALS). Mutations in more than 30 different genes cause fALS, most prominently those encoding C9orf72, Superoxide Dismutase 1 (SOD1), Fused in sarcoma (FUS) and Tar DNA-binding protein 43 (TDP-43) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In most ALS cases, TDP-43 [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and, less frequently FUS [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], mis-localize from the nucleus to the cytosol where they accumulate and aggregate. The prion-like and highly intrinsically disordered domains of FUS and TDP-43 predispose these proteins to mis-localization and misfolding [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Also, both proteins form condensates by liquid-liquid phase separation [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which may promote conversion of the accumulated cytosolic misfolded proteins into more stable, insoluble aggregates in the neurons of ALS patients. Of note, the role of these aggregates in ALS pathogenesis remains unclear.\u003c/p\u003e \u003cp\u003eKey characteristics of TDP-43 and FUS, such as their misfolding, mis-localization, accumulation, and the ensuing cytotoxicity have been recapitulated in yeast models [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These yeast models identified genetic modulators of TDP-43 [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and FUS [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] toxicity, leading to the discovery of modulators of ALS [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Yeast models have also been instrumental in deciphering the molecular and cellular mechanisms underlying TDP-43 and FUS aggregation [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the occurrence of protein aggregates in brain and spinal cord tissues are well-established neuropathological hallmarks of ALS/FTD, the causative link between these aggregates and disease progression remains tenuous. Numerous studies indicate only weak correlations between aggregation and eventual neuronal cell death [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Conversely, many findings even indicate potential protective functions of protein aggregates in diverse neurodegenerative diseases [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Accordingly, genetic modifiers of the toxicity of misfolded proteins are not necessarily modifiers of their aggregation [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although TDP-43 and FUS aggregation is a clear indicator of impaired proteostasis, it remains unclear exactly how TDP-43 and FUS aggregates contribute to cellular toxicity, if at all.\u003c/p\u003e \u003cp\u003eMoreover, the role of ageing in the formation and persistence of protein aggregates and toxicity of misfolded proteins has been notoriously difficult to ascertain. Indeed, chronological ageing hampers the ability of cells to maintain proteostasis [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, the inability to form and maintain protein aggregates in aged cells is associated with reduced lifespan [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], indicating that age-dependent protein aggregates can be protective. While cultured mammalian cells are often used to model cellular senescence [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], they do not entirely recapitulate most physiological aspects of ageing. Studies of TDP-43 and FUS misfolding in the context of ageing have relied heavily on rodent models [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which are too complex to track cellular and molecular details linking ageing to TDP-43 and FUS aggregation and toxicity.\u003c/p\u003e \u003cp\u003eIn addition to impaired proteostasis, ageing cells must contend with errors in protein biosynthesis, or mistranslation. Many have speculated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and shown experimentally [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] that mistranslation adversely affects longevity. While the specific mechanisms by which translation fidelity influences lifespan remain unclear, mistranslation clearly impairs proteostasis and induces protein misfolding and aggregation [\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This plausibly contributes to the decline of proteostasis seen in ageing cells. Further, mistranslation modulates the misfolding, aggregation and toxicity of neurodegenerative disease-associated proteins, such as polyglutamine expanded huntingtin and FUS [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, the degree to which ageing and mistranslation synergize to affect the misfolding of the ALS/FTD-associated proteins FUS and TDP-43 remains enigmatic.\u003c/p\u003e \u003cp\u003eYeast models have a rich history in the study of ageing, including the identification of key cellular factors that influence longevity, such as TOR kinase complex 1 (TOR) signaling [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], the unfolded protein response [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], reactive oxygen species[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and metabolism [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Additionally, yeast models have been employed to study how translation errors influence cellular fitness and proteostasis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Yet, the combination of ageing and mistranslation has not yet been studied in yeast models of misfolded proteins, including TDP-43 and FUS.\u003c/p\u003e \u003cp\u003eTo determine how ageing modulates the aggregation and toxicity of FUS and TDP-43, we establish optimized yeast models with low expression of FUS and TDP-43. These models allowed us to determine that mitochondrial respiration and β-oxidation enhances FUS and TDP-43 toxicity, but not necessarily their aggregation. Furthermore, we confirm that ageing impairs proteostasis, as evidenced by increased aggregation of a metastable reporter proteins. By contrast, ageing antagonizes FUS and TDP-43 aggregation while exacerbating the growth defect caused by FUS and TDP-43. Finally, we find that further impairing proteostasis in ageing cells by inducing translation errors synergistically prevents FUS and TDP-43 aggregation and unmask toxic phenotypes associated with FUS and TDP-43. Accordingly, our findings suggest that the loss of proteostatic capacity in ageing cells reduces aggregation of FUS and TDP-43 and thus increases their toxicity. Our findings contribute to the growing literature indicating that protein aggregates are not necessarily the major toxic species in neurodegenerative diseases, highlighting the need to investigate misfolded monomer and oligomer species as the likely culprits. Finally, our findings genetically identify translation errors, such as those caused by tRNA variants, as potential contributors or risk factors for ALS/FTD and other age-associated protein misfolding diseases.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cem\u003eRespiring cells are sensitive to FUS and TDP-43.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eYeast models of FUS and TDP-43 aggregation and toxicity have been useful discovery tools for screening genetic interactors that antagonize toxicity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the high degree of toxicity caused by high expression levels of FUS and TDP-43 in these models has made identifying potential enhancers of toxicity challenging. To this end, we devised models that express low levels of FUS and TDP-43 using the MET17 promoter.\u003c/p\u003e \u003cp\u003eWe compared yeast models expressing high levels of FUS and TDP-43 regulated by the common GAL1 promoter to our models expressing lower levels of FUS and TDP-43 regulated by the MET17 promoter. The MET17 promoter induced lower steady-state levels of both FUS and TDP-43 when compared to the GAL1 promoter, which corresponded with a milder growth defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-F).\u003c/p\u003e \u003cp\u003eNeurons are uniquely dependent on oxidative mechanisms of producing ATP for energy [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Yet, whether mitochondrial respiration influences the sensitivity of cells to toxicity of FUS and TDP-43 is unclear. Foregoing the GAL1 promoter in favor of the MET17 promoter enabled us to test how mitochondrial respiration influences the fitness defects caused by FUS and TDP-43. When exposed to fermentable carbon sources (glucose), yeast cells predominantly generate energy by glycolysis [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Instead, when forced to metabolize non-fermentable carbon sources, yeast cells generate energy by mitochondrial respiration [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. We took advantage of the ease with which the metabolism of yeast can be manipulated to study how obligately respiring cells manage the misfolding and fitness defects associated with FUS and TDP-43. We compared cells producing energy via glycolysis (glucose) to those generating energy by oxidative phosphorylation (glycerol and potassium acetate) and β-oxidation or short (myristic acid) and long (oleic acid) chain fatty acids [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The fitness defects caused by FUS and FUS-YFP are exacerbated in cells grown on plates supplemented with glycerol, potassium acetate, myristic acid and oleic acid when compared to cells grown on plates supplemented with glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Similarly, the fitness defect caused by TDP-43 is exacerbated by growth on glycerol, potassium acetate and myristic acid, but not oleic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). We next tested whether the extent of FUS and TDP-43 aggregation depends on metabolic program. We confirmed that expression of FUS-YFP and TDP43-YFP induces the formation of both FUS-YFP and TDP43-YFP foci in cells grown in glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-L). Aggregation of FUS-YFP was impaired in cells grown in media containing glycerol, potassium acetate and oleic acid, while myristic acid increased the number of FUS-YFP aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). TDP43-YFP aggregation was reduced in cells grown in media containing potassium acetate, while both myristic acid and oleic acid increased the number of TDP43-YFP aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L).\u003c/p\u003e \u003cp\u003eIn sum, we generated a low expression FUS and TDP-43 yeast model. Using our novel model, we demonstrate that altering metabolic program changes the extent of aggregation and the fitness defects caused by FUS and TDP-43.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIncreased FUS and TDP43 toxicity in chronologically aged cells\u003c/h2\u003e \u003cp\u003eAs post-mitotic cells, neurons must maintain proteostasis despite an accumulation of damage throughout their lifespan. Although protein aggregation is strongly associated with advanced age [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], our options for monitoring protein aggregation in ageing mammalian cells are limited. Model organisms provide an effective alternative to study proteostasis and disease-associated protein aggregation in ageing cells. Unlike neurons, yeast cells age through two mechanisms: chronological and replicative ageing [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Chronological ageing, which describes the lifespan of a yeast cell stationary phase [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], more accurately recapitulates ageing experienced by neurons than replicative ageing, which describes the number of divisions a mother cell can sustain before senescence [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Using our low expression FUS and TDP-43 yeast models, we investigated whether FUS and TDP-43 exacerbate the toxicity associated with chronological ageing.\u003c/p\u003e \u003cp\u003eWe first tested if ageing cells are sensitive to FUS and TDP-43 misfolding. We performed regrowth assays using cells expressing low levels of FUS or FUS-YFP that were aged for eight days. Both FUS and FUS-YFP induced a growth defect in young cells (two days of ageing) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). FUS and FUS-YFP also reduced the ability of cells to regrow after four and eight days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We then aged cells expressing FUS or FUS-YFP for eight days in caloric restriction media (0.2% glucose), which extends lifespan in yeast [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and monitored the ability of these cells to regrow on standard media (2% glucose). While both FUS and FUS-YFP still induced a growth defect in young cells (two days of ageing) grown in caloric restriction media, eight days of ageing did not exacerbate this growth defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We next performed regrowth assays with cells expressing low levels of TDP-43 and aged for a time course of eight days. TDP-43 induced a growth defect in young cells, and reduced the ability of cells to regrow after four and eight days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The growth defect associated with TDP-43 was exacerbated after four days of ageing in caloric restriction media but was not exacerbated after eight days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eWe next tested whether FUS and TDP-43 exacerbated cell death associated with ageing. Using propidium iodide staining as a proxy for cell death, we measured the percentage of dead cells in control cells and cells expressing FUS or TDP-43 after two, four and eight days of ageing. Expression of FUS did not change the percentage of dead cells when compared to control cells at any point of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In contrast, TDP-43 mildly reduced the percentage of dead cells when compared to control cells after eight days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Ageing cells in caloric restriction media reduced the percentage of dead cells after eight days of ageing, regardless of the expression of FUS or TDP-43 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003eWe performed western blots to test whether ageing changes steady state levels of FUS and TDP-43. Steady state levels of FUS and TDP-43 did not change when cells were aged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). We then turned our attention to how FUS and TDP-43 influence expression of molecular chaperones in ageing cells. We found that expression of FUS and TDP-43 reduced steady state levels of Hsp104, Hsp42 and Hsp26 when compared to control cells after two days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-K). Cells expressing FUS maintained lower levels of Hsp104 and Hsp42 after four days of ageing, while Hsp26 levels recovered back to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-K). In contrast, cells expressing TDP-43 maintained lower levels of Hsp42 and Hsp26, while Hsp104 levels recovered back to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-K).\u003c/p\u003e \u003cp\u003eIn sum, both FUS and TDP-43 impair the fitness of aged cells, although neither FUS nor TDP-43 exacerbate cell death caused by ageing. Furthermore, both FUS and TDP-43 reduce steady state levels of Hsp104, Hsp42 and Hsp26, possibly further impairing proteostasis in ageing cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAgeing reduces FUS and TDP-43 inclusions.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eNeurodegenerative phenotypes have long been associated with aggregation of misfolded proteins, like FUS and TDP-43, in ageing neurons. We sought to unravel the relationship between chronological ageing and aggregation of FUS and TDP-43. To this end, we monitored aggregation of FUS-YFP and TDP43-YFP during the aging process.\u003c/p\u003e \u003cp\u003eWe aged cells expressing FUS-YFP in standard media (2% glucose) and caloric restriction media (0.2% glucose) for a time course of four days. Both the percentage of cells with FUS-YFP foci and the number of FUS-YFP foci per cell were reduced after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Caloric restriction also reduced the percentage of cells with FUS-YFP foci but masked the reduction in FUS-YFP foci caused by ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Although low levels of FUS does not induce cell death, we hypothesized that the ability of cells to maintain FUS aggregates may influence the population of cells that die. To test this, we next stained cells expressing FUS-YFP with propidium iodide to assess the occurrence of FUS-YFP inclusions in dying cells. Across all conditions, cells stained with propidium iodide less frequently contained FUS-YFP foci when compared to their unstained counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003eWe next aged cells expressing TDP43-YFP for four days. Similarly to FUS-YFP, both the percentage of cells with TDP43-YFP foci and the number of TDP43-YFP foci per cell were reduced after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). In contrast to FUS-YFP, the percentage of cells with TDP43-YFP foci was not reduced by caloric restriction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). In addition, when monitoring cells stained with propidium iodide, we find that cells stained with propidium iodide less frequently contained TDP43-YFP foci when compared to their unstained counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003eTo confirm that ageing impairs proteostasis, we aged cells expressing metastable Firefly Luciferase-GFP (FFL-GFP) as proteostasis reporter that is not directly linked to neurodegenerative diseases. The percentage of cells with FFL-GFP foci and the number of FFL-GFP foci per cell increased after four days of ageing (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C). To assess whether caloric restriction influences proteostasis decline in ageing cells, we aged cells expressing FFL-GFP in caloric restriction media. Caloric restriction did not change the percentage of cells with FFL-GFP foci, nor the number of FFL-GFP foci per cell (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C). As dead cells lose the ability to maintain FUS and TDP-43 inclusions, we sought to assess whether they also lose the ability to maintain FFL-GFP inclusions. Similar to FUS-YFP and TDP43-YFP, cells stained with propidium iodide showed a drastic reduction in the percentage of cells with FFL-GFP foci (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003eIn sum, our findings indicate a reduction in both FUS and TDP-43 aggregation in ageing cells. In contrast, FFL aggregation increases in ageing cells, both showing specificity and confirming impaired proteostasis in ageing cells. Finally, our findings reveal a negative correlation between FUS, TDP-43 and FFL aggregation and cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFUS and TDP-43 disrupt molecular chaperone solubility.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eMolecular chaperones are major modulators of protein folding and aggregation [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In particular, the AAA-ATPase disaggregase Hsp104 and the holdases Hsp42 and Hsp26 cooperate to regulate protein aggregation in yeast cells. Importantly, loss of Hsp104, Hsp42 and Hsp26 impairs fitness of ageing cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. We explored whether FUS and TDP-43 dysregulate the function of Hsp104, Hsp42 and Hsp26 in ageing cells, leading to loss of aggregation.\u003c/p\u003e \u003cp\u003eFirst, we monitored the solubility of FUS and TDP-43 to confirm loss of FUS and TDP-43 aggregation in ageing cells. We performed sedimentation assays using ageing cells expressing either FUS or TDP-43 to separate proteins into soluble (s) and insoluble (p) fractions. Both FUS and TDP-43 were less present in the insoluble fraction, and thus more soluble, after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eWe next turned our attention to the solubility of Hsp104, Hsp42 and Hsp26 in ageing cells expressing FUS and TDP-43. Expression of FUS, but not TDP-43, reduced the solubility of Hsp104 when compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G). In both control cells and, to a lesser extent, cells expressing FUS, Hsp104 became more soluble after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). However, in cells expressing TDP-43, Hsp104 became less soluble after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G). Both FUS and TDP-43 reduced the solubility of Hsp42 when compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). While Hsp42 became less soluble after four days of ageing in control cells, Hsp42 became more soluble after four days of ageing in cells expressing FUS, but not TDP-43 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). In similar fashion to Hsp42, both FUS and TDP-43 reduced the solubility of Hsp26 when compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). In both control cells and, to a lesser extent, cells expressing FUS, Hsp26 became less soluble after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). In contrast, Hsp26 solubility did not change in cells expressing TDP-43 after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eWe next monitored FUS-mCherry and TDP43-mCherry aggregation in cells deleted for Hsp104 (\u003cem\u003eΔhsp104\u003c/em\u003e) or Hsp42 and Hsp26 (\u003cem\u003eΔhsp42Δhsp26\u003c/em\u003e). Loss of Hsp104 reduced the percentage of cells with FUS-mCherry and TDP43-mCherry foci and the number of FUS-mCherry and TDP43-mCherry foci per cell (Fig. S2A-C). However, loss of Hsp42 and Hsp26 only reduced the percentage of cells with TDP43-mCherry foci and the number of TDP43-mCherry foci per cell (Fig. S2A-C). We also performed growth assays to determine whether loss of FUS and TDP-43 aggregation affects growth. Loss of Hsp104 did not change the growth of cells expressing FUS or TDP-43 (Fig. S2D-E). However, loss of Hsp42 and Hsp26 exacerbated the growth defect caused by FUS, but rescued the growth defect caused by TDP-43.\u003c/p\u003e \u003cp\u003eAs both FUS and TDP-43 modulate the solubility of molecular chaperones, we tested whether FUS and TDP-43 impair proteostasis in ageing cells. We expressed a metastable FFL-mCherry reporter in ageing cells co-expressing FUS and TDP-43. FUS and TDP-43 do not increase the percentage of cells with FFL-mCherry foci, nor the number of FFL-mCherry foci per cell in young cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-L). However, after four days of ageing, FUS increased the number of FFL-mCherry foci per cell when compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). In stark contrast, TDP-43 decreased both the percentage of cells with FFL-mCherry foci and the number of FFL-mCherry foci per cell when compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-L).\u003c/p\u003e \u003cp\u003eIn sum, both FUS and TDP-43 reduce the solubility of the molecular chaperones Hsp104, Hsp42 and Hsp26 in ageing cells. Furthermore, loss of Hsp104, Hsp42 and Hsp26 function impairs aggregation of FUS and TDP-43, indicating a potential mechanism underlying the reduced aggregation of FUS and TDP-43 in ageing cells. Finally, although both FUS and TDP-43 reduce solubility of molecular chaperones, they have different effects on protein folding, as measured by FFL-mCherry aggregation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eMistranslation unmasks toxicity of FUS and TDP-43 in ageing cells.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe ability of organisms to maintain translation fidelity is strongly linked to increased longevity [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Inducing translation errors (mistranslation) is detrimental to proteostasis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], and can even modulate aggregation of endogenous proteins [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], metastable proteins [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and proteins associated with neurodegenerative diseases like polyglutamine expanded huntingtin protein [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and FUS [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, how mistranslation impacts proteostasis in ageing cells is poorly understood.\u003c/p\u003e \u003cp\u003eWe sought to investigate whether mistranslating tRNA variants affect chronological lifespan of cells. To induce mistranslation, we expressed serine tRNA variants with anticodon mutations that induce proline to serine misincorporation at rates of approximately 0.5% (P\u0026thinsp;\u0026gt;\u0026thinsp;S weak) or 5% (P\u0026thinsp;\u0026gt;\u0026thinsp;S strong), or arginine to serine misincorporation at a rate of approximately 3.5% (R\u0026thinsp;\u0026gt;\u0026thinsp;S) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. We performed growth assays using cells expressing mistranslating tRNA variants and aged over eight days in either standard media (2% glucose) or caloric restriction media (0.2% glucose). P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S both impair the ability of cells to regrow after eight days of ageing in standard media, but not caloric restriction media (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). We stained mistranslating cells with propidium iodide to measure the percentage of dead cells. While mistranslating tRNA variants did not exacerbate cell death in cells aged in standard media, P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S partially antagonized the protective effects of caloric restriction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003eWe explored whether mistranslating tRNA variants augment the toxicity associated with FUS and TDP-43 in ageing cells. We aged cells co-expressing mistranslating tRNA variants and either FUS or TDP-43 for eight days. P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S synergistically impaired the ability of cells expressing FUS and TDP-43 to regrow after eight days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S also induces synthetic cell death in cells expressing FUS and TDP-43 after four and eight days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-H).\u003c/p\u003e \u003cp\u003eIn sum, mistranslating tRNA variants impair the fitness of ageing cells, but they do not exacerbate cell death when expressed in isolation in ageing cells. However, when expressed in addition to FUS and TDP-43, mistranslation synergistically impairs fitness of ageing cells by inducing synthetic cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eMistranslation reduces FUS and TDP-43 inclusions in ageing cells.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThus far, we have demonstrated an inverse correlation between ageing and inclusion formation of FUS and TDP-43, indicating a potential protective function of FUS and TDP-43 inclusions. We speculated that the synergistic toxicity of mistranslating tRNA variants and FUS and TDP-43 in ageing cells is due to an inability of cells to form protective inclusion of FUS and TDP-43. To test this hypothesis, we monitored FUS-YFP and TDP43-YFP aggregation in ageing cells expressing mistranslating tRNA variants.\u003c/p\u003e \u003cp\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;S weak, P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S all reduced the percentage of cells containing FUS-YFP foci, while P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S reduced the number of FUS-YFP foci per cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). After ageing cells for four days, P\u0026thinsp;\u0026gt;\u0026thinsp;S weak, P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S led to a greater decrease in the percentage of cells with FUS-YFP foci and the number of FUS-YFP foci per cell when compared to cells expressing wt tSer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). While mistranslating tRNA variants had no effect on the percentage of cells with TDP43-YFP foci, P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S both reduced the number of TDP43-YFP foci per cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). Although four days of ageing reduced both the percentage of cells with TDP43-YFP foci and the number of TDP43-YFP foci per cell regardless of the tRNA expressed, mistranslating tRNA variants did not change the percentage of cells with TDP43-YFP foci and the number of TDP43-YFP foci per cell after four days of ageing (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003eWe used the metastable FFL-GFP reporter to test whether mistranslating tRNA variants reduces aggregation of FUS and TDP-43 by impairing proteostasis. P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S both increased the percentage of cells containing FFL-GFP foci, but not the number of FFL-GFP foci per cell in young cells (Fig. S3A-C). After ageing cells for four days, P\u0026thinsp;\u0026gt;\u0026thinsp;S strong and R\u0026thinsp;\u0026gt;\u0026thinsp;S both led to a greater increase in the number of FFL-GFP foci per cell, but not the percentage of cells containing FFL-GFP foci, when compared to cells expressing wt tSer.\u003c/p\u003e \u003cp\u003eIn sum, our findings suggest that mistranslating tRNA variants reduce aggregation of FUS and TDP-43 in both young and aged cells, supporting our hypothesis that FUS and TDP-43 inclusions are protective. In contrast, mistranslating tRNA variants increase aggregation of FFL-GFP in young and aged cells, confirming that mistranslation impairs proteostasis and indicating that impaired proteostasis inhibits aggregation of FUS and TDP-43.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eLike many other neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS) are associated with the accumulation of aggregated proteins, such as Fused in sarcoma (FUS) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and Tar DNA-binding protein 42 (TDP-43) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Accordingly, protein aggregates had been proposed to act as key drivers of neurodegeneration and age-related cell death, generally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. However, many detailed studies challenge the notion that protein aggregation causes toxicity in models of ageing and neurodegeneration [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, fidelity of protein biosynthesis is clearly a modifier of ageing [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and protein aggregation [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], including aggregation of proteins associated with neurodegenerative diseases [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Yeast has been an effective model to study chronological ageing [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], translation errors [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], and aggregation of neurodegenerative disease-associated proteins [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Here, we explore the combined influence of ageing and translation errors on aggregation of FUS and TDP-43 and their toxicity, which closely reflects the accumulation of different proteostasis stressors experienced over the lifespan of neurons in ALS.\u003c/p\u003e \u003cp\u003ePreviously described FUS and TDP-43 yeast models expressed very high levels of FUS and TDP-43 [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which have delivered important insights into aggregation and cytotoxicity of FUS and TDP-43. However, owing to their acute cytotoxicity, these models are not optimal for investigating metabolism and chronological ageing as modifiers of FUS and TDP-43 aggregation and their toxicity. We overcome these limitations by devising yeast models that express low levels of FUS and TDP-43, resulting only mild growth defects. Furthermore, our optimized yeast models facilitate studies in media containing non-fermentable carbon sources, driving yeast cells to energy production via mitochondrial respiration [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], which mimics the metabolic activity in neurons [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. We show that cells producing energy by oxidative phosphorylation or β-oxidation are sensitized to expression of both FUS and TDP-43 compared to fermenting yeast. Notably, respiring cells frequently show a reduction in FUS and TDP-43 aggregates, suggesting an uncoupling of FUS and TDP-43 aggregation from their cytotoxicity. Therefore, we speculate that aggregation is not necessarily the key mechanism by which FUS and TDP-43 induce toxicity in respiring cells.\u003c/p\u003e \u003cp\u003eOur study further explores how chronological ageing alters the accumulation of FUS and TDP-43 aggregates and their toxicity. When cells are aged chronologically, we find that FUS and TDP-43 do not induce cell death, but instead prevent aged cells from re-entering the cell cycle. Further, we reveal a negative correlation between ageing and FUS and TDP-43 aggregation. Notably, protein aggregation was not universally inhibited by ageing, as ageing increased the aggregation of a commonly used metastable reporter protein, Firefly Luciferase-GFP (FFL-GFP). These findings may reveal a specific impact of ageing on the RNA granule condensates that TDP-43 and FUS associate with[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] compared to the spatial quality control compartments that aggregated FFL is sequestered to [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Moreover, cells that die during the ageing process consistently retain fewer FUS and TDP-43 aggregates compared to their surviving counterparts. We propose that FUS and TDP-43 aggregates perform protective functions, and that their presence may be predictive of cell survival. Accordingly, we postulate that ageing cells are particularly sensitive to the misfolding of FUS and TDP-43, plausibly due to impaired proteostasis[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] an inability to form or maintain protective aggregates [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We also assessed whether FUS and TDP-43 interact with proteostasis networks to impair their aggregation in ageing cells. Indeed, FUS and TDP-43 reduce the steady-state levels and the solubility of molecular chaperones, thereby likely reducing their protective functions in proteostasis and compromising longevity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan additionalcitationids=\"CR73 CR74 CR75\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe furthermore investigate whether mistranslating tRNA variants exacerbate ageing phenotypes in our FUS and TDP-43 yeast models. Our findings indicate that, while mistranslating tRNA variants do not induce cell death, they prevent aged cells form re-entering the cell cycle. However, mistranslating tRNA variants partially reduce the protective effect of caloric restriction on age-related cell death. Remarkably, although neither mistranslating tRNA variants, nor FUS and TDP-43, exacerbate age-related cell death, the combination of mistranslation and FUS or TDP-43 induces synthetic toxicity in ageing cells. Notably, the stress caused by mistranslating tRNA variants closely mimics that of ageing, impairing proteostasis and increasing aggregation of FFL-GFP, while antagonizing the aggregation of FUS and TDP-43. We deduce that ageing, mistranslation and expression of FUS or TDP-43 synergistically impair proteostasis, preventing formation of protective FUS and TDP-43 aggregates and foiling cellular survival. These findings support our hypothesis that the inability to maintain FUS and TDP-43 aggregates in ageing cells promotes their toxicity.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eMost ALS patients accumulate aggregated proteins, like TDP-43 and FUS, in their neurons. The mechanism by which ageing modulates this aggregation, and whether these aggregates cause disease has remained elusive. Here, we find that ageing, paradoxically but specifically, impairs aggregation of FUS and TDP-43, plausibly due to impaired proteostasis. We also highlight mistranslation as a modulator of FUS and TDP-43 toxicity in ageing cells. Based on our findings, we propose a model, whereby mistranslation and possibly other challenges to proteostasis, combined with ageing impair aggregation, thus unleashing the cytotoxicity associated with smaller, more soluble misfolded FUS and TDP-43 conformers. Our research underscores the need to determine the molecular and cellular mechanisms underlying protective protein aggregation and explore if this model also to other misfolded proteins and neurodegenerative diseases.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids\u003c/h2\u003e \u003cp\u003eAll plasmids used in this study are described in Supplementary Table\u0026nbsp;1. The plasmids encoding TDP-43, FUS and FUS-YFP under control of the MET17 promoter were generated using cut-paste cloning by Analisa Echeverria (this study). The plasmid encoding TDP43-YFP was generated using Gateway cloning. The plasmid encoding FUS under control of the GAL1 promoter was a kind gift from Dr. Aaron Gitler [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The plasmid encoding TDP-43 under control of the GAL1 promoter was generated by Gateway cloning. The p426MET17 FFL\u003csub\u003eD50N,G119N\u003c/sub\u003e-GFP plasmid was a kind gift from Dr. John Glover [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The plasmids encoding TDP-43, FUS and FFL\u003csub\u003eD50N,G119N\u003c/sub\u003e-mCherry were all generated using Gateway cloning using a pAG416GPD ccdB mCherry destination vector, which was designed using cut-paste cloning. Plasmids encoding tRNA variants were a kind gift from Dr. Chris Brandl (pSUP17-wt, pSUP17-P\u0026thinsp;\u0026gt;\u0026thinsp;S\u003csub\u003eweak\u003c/sub\u003e, pSUP17-P\u0026thinsp;\u0026gt;\u0026thinsp;S\u003csub\u003estrong\u003c/sub\u003e, pSUP17-R\u0026thinsp;\u0026gt;\u0026thinsp;S) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eYeast strains, media and culturing conditions\u003c/h2\u003e \u003cp\u003eAll experiments were performed using derivatives of the W303 yeast strain (\u003cem\u003eleu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15\u003c/em\u003e). Strains deleted for HSP104, or HSP42 and HSP26 were described previously [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Yeast cells were transformed with desired plasmids encoding either FUS, TDP-43 or FFL\u003csub\u003eD50N,G119N\u003c/sub\u003e using the standard LiAc/PEG protocol [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Cells were cultured in and plated on agar plates containing synthetic defined (SD) media supplemented with 2% glucose, 6.7 g/mL yeast nitrogenous bases with ammonium sulfate and amino acids (40 mg/mL L-lysine, 20 mg/mL L-arginine, 10 mg/mL L-threonine, 60 mg/mL L-phenylalanine, 20 mg/mL L-isoleucine, 10 mg/mL L-methionine, 20 mg/mL adenine hemisulfate). Media was supplemented with additional amino acids for selection where necessary (20 mg/mL L-histidine, 60 mg/mL L-leucine, 20 mg/mL uracil, 80 mg/mL L-tryptophan). L-methionine was omitted from the media when necessary for induction of the MET17 promoter. For ageing experiments, cells were grown to saturation in a 3mL liquid culture for 24 hours before cells were pelleted and resuspended in media lacking methionine. Cells were then allowed to age chronologically for eight days. At the indicated time points, aliquots of the culture were taken to perform experiments. For caloric restriction, cells were grown to saturation in a 3 mL liquid culture for 24 hours before the cells were pelleted and resuspended in media lacking methionine and containing only 0.2% glucose.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowth assay\u003c/h3\u003e\n\u003cp\u003eTo assess growth defects, we performed growth assays as described by Petropavlovskiy et al. [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Briefly, cells were diluted to OD600\u0026thinsp;=\u0026thinsp;1 and 5-fold serial dilutions were performed of each sample in a 96-well plate. Using a 48-pronged frogger, single spots of each sample were plated onto SD agar plates containing 2% glucose. When stated, cells were plated on agar plates that were modified to contain different carbon sources (2% galactose, 2% glycerol, 2% potassium acetate (KoAc), 0.1% myristic acid solubilized in 0.05% Tween20, or 0.1% oleic acid solubilized in 0.05% Tween20) instead of 2% glucose. Plates were incubated at 30\u0026deg;C for 24\u0026ndash;72 hours before plates were photographed and analyzed using ImageJ. The densitometry of the spots at the same dilution factor were later compared. Experiments were repeated at least three times with independent transformants.\u003c/p\u003e\n\u003ch3\u003ePropidium iodide staining and flow cytometry\u003c/h3\u003e\n\u003cp\u003eTo assess cell death, we performed propidium iodide staining as described by Chadwick et al. [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Briefly, 100 \u0026micro;L of cell culture was aliquoted into a labelled tube, pelleted and resuspended in 100 \u0026micro;L of 5 \u0026micro;g/mL propidium iodide in PBS. After 10 minutes, the cells were pelleted and resuspended in PBS. The percentage of propidium iodide-stained cells was determined by flow cytometry using the BD Bioscience FACS Celesta flow cytometer. A 561 nm laser was used to identify cells stained with propidium iodide. A sample of cells that were boiled for five minutes was used to generate a gate corresponding to propidium iodide positive staining. At least three independent experiments were performed for each sample, with a minimum of 150,000 cells being analyzed.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence microscopy\u003c/h2\u003e \u003cp\u003eTo measure the formation of protein inclusions, we performed fluorescence microscopy using cells expressing either FFL-GFP or FUS-YFP. Briefly, 100 \u0026micro;L of cell culture was aliquoted into a labelled tube and stained with propidium iodide as previously described. Cells were then imaged using the BioTek Cytation 5 Cell Imaging Multi-mode Reader at 20x magnification. Images were taken using a GFP filter to visualize and assess inclusion formation, and a TexasRed filter to visualize and assess cells stained with propidium iodide. Experiments were repeated at least three times with independent transformants and at least 400 cells were imaged and later analyzed. Downstream analysis of fluorescence microscopy images was performed in R using the EBImage package in R [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and Western Blot\u003c/h2\u003e \u003cp\u003eTo measure steady state levels of proteins, protein extraction was performed using a modified alkaline lysis protocol [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Briefly, 0.5 mL of OD600\u0026thinsp;=\u0026thinsp;2 cells were harvested and resuspended in 50 \u0026micro;L alkaline lysis buffer (0.1M NaOH, 2% SDS, 50mM EDTA) and boiled for five minutes. The lysate was then cleared at 21k xg for 10 minutes, transferred to a fresh tube and diluted in loading buffer and boiled again for five minutes. Protein lysates were then resolved by SDS-PAGE and transferred to PVDF membrane followed by immunoblotting with anti-FUS (Bethyl, A300-302A, 1:500), anti-TDP-43 (Novus, H00023435-M01, 1:500), anti-Pgk1 (Origene, AP21371AF-N, 1:5000), anti-Hsp104, anti-Hsp42 and anti-Hsp26 (all kind gifts from Dr. Johannes Buchner). Secondary antibodies used were either conjugated with Alexa680 (Thermofisher, 1:5000) or HRP (Thermofisher, 1:5000). Blots were imaged and the densitometry of bands was assessed and compared in relation to the Pgk1 loading control. Experiments were repeated at least three times with independent transformants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSedimentation assay\u003c/h2\u003e \u003cp\u003eTo assess solubility of proteins, a mild mechanical lysis was used to extract proteins. Briefly, 3 mL of OD600\u0026thinsp;=\u0026thinsp;2 cells were harvested and resuspended in 100 \u0026micro;L mild lysis buffer (100 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM DTT, 5 mM EDTA, 50 \u0026micro;M NEM and 10 \u0026micro;M PMSF). Cell suspension was then transferred to an Eppendorf tube containing 100 \u0026micro;L acid-washed glass beads (Sigma, G8772) and then disrupted for five 1-minute cycles interspersed by 1-minute incubations on ice. The lysate was then cleared at 200 xg for 2 minutes before being transferred to a new tube. An aliquot of the total lysate was taken before the remaining lysate was centrifuged at 500 xg for 15 minutes. The supernatant was aliquoted into a new tube and the pellet was then resuspended in mild lysis buffer. All protein fractions were diluted with an equal volume of SUMEB (1% SDS, 8 M Urea, 10 mM MOPS, 10 mM EDTA). Equal volumes of each protein fraction were then resolved by SDS-PAGE followed by immunoblotting as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll graphical representations of data and statistical analysis were performed using GraphPad Prism. Data were compared using either one or two-way ANOVA followed by Fisher\u0026rsquo;s LSD. All relevant p-values are stated.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eWe have no competing interests to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDonovan W. McDonald: Investigation, Conceptualization, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing\u0026mdash;original draft, Writing\u0026mdash;review \u0026amp; editing. Nikita Chugh: Investigation, Formal analysis. Rares Sava: Investigation, Formal analysis. Martin L. Duennwald: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing\u0026mdash;review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Analisa Echeverria (Boston Biomedical) for generating the MET17 driven TDP-43, FUS and FUS-YFP plasmids. We would also like to thank Drs. Aaron D. Gitler (Stanford University), John R. Glover (University of Toronto) and Christopher J. Brandl (University of Western Ontario, Emeritus) for providing plasmids used in this study. We would also like to thank Drs. Patrick Lajoie (University of Western Ontario) and Emily M. Sontag (Marquette University) for their critical editing of this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The R script used to analyze fluorescence microscopy is available at https://doi.org/10.6084/m9.figshare.28688966.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNeumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al. Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Science (1979). 2006;314:130\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eArai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H, et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. 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BIOINFORMATICS APPLICATIONS NOTE. 2010;26:979\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003evon der Haar T. Optimized Protein Extraction for Quantitative Proteomics of Yeasts. PLoS One. 2007;2:e1078.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Protein aggregation, protein misfolding, molecular chaperone, ageing, mitochondria, mistranslation, ALS, TDP-43, FUS","lastPublishedDoi":"10.21203/rs.3.rs-6448360/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6448360/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein aggregation is indicative of the loss of proteostasis associated with neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Proteins like Fused in sarcoma (FUS) and Tar DNA-binding protein 43 (TDP-43) accumulate and aggregate in the cytosol of neurons in ALS/FTD. Yet, it remains unclear how ageing affects FUS and TDP-43 aggregation, and how these aggregates in turn influence neurodegeneration in ALS/FTD. In addition, mistranslation can reduce longevity, challenge proteostasis, and modulate protein aggregation. To investigate how ageing and mistranslation modulate FUS and TDP-43 aggregation and toxicity, we enlist tractable and reliable yeast models. We establish that respiring yeast cells are sensitized to FUS and TDP-43 compared fermenting cells, yet this increased sensitivity does not correlate with increased FUS and TDP-43 aggregation. We also demonstrate that chronological ageing and mistranslation caused by tRNA variants antagonize FUS and TDP-43 aggregation and synergize to exacerbate FUS and TDP-43 cytotoxicity. Our work provides new insights into factors that uncouple FUS and TDP-43 aggregation from toxicity and support a rather protective role for FUS and TDP-43 aggregates in promoting longevity.\u003c/p\u003e","manuscriptTitle":"Mistranslation and ageing unmask FUS and TDP-43 toxicity in yeast models of neurodegenerative diseases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 16:05:05","doi":"10.21203/rs.3.rs-6448360/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-25T13:26:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-08T14:41:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80883444945095160762721651650694718442","date":"2025-05-30T19:42:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-21T06:02:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91320464796072344541325762296711516922","date":"2025-05-11T06:34:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T10:11:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-09T08:21:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-08T16:10:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biology","date":"2025-05-07T18:38:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"21d4d59a-1199-4017-8d08-a212c4702fc6","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-27T11:26:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 16:05:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6448360","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6448360","identity":"rs-6448360","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}