A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

Expression of Wild-Type and Mutant Human TDP-43 in Yeast Inhibits TOROID (TORC1 Organized in Inhibited Domain) Formation and Autophagy Proportionally to the Levels of TDP-43 Toxicity.

TDP-43 forms aggregates in the neurons of patients with several neurodegenerative diseases. Human TDP-43 also aggregates and is toxic in yeast. Here, we used a yeast model to investigate (1) the nature of TDP-43 aggregates and (2) the mechanism of TDP-43 toxicity. Thioflavin T, which stains amyloid but not wild-type TDP-43 aggregates, also did not stain mutant TDP-43 aggregates made from TDP-43 with intragenic mutations that increase or decrease its toxicity. However, 1,6-hexanediol, which dissolves liquid droplets, dissolved wild-type or mutant TDP-43 aggregates. To investigate the mechanism of TDP-43 toxicity, the effects of TDP-43 mutations on the autophagy of the GFP-ATG8 reporter were examined. Mutations in TDP-43 that enhance its toxicity, but not mutations that reduce its toxicity, caused a larger reduction in autophagy. TOROID formation, which enhances autophagy, was scored as GFP-TOR1 aggregation. TDP-43 inhibited TOROID formation. TORC1 bound to both toxic and non-toxic TDP-43, and to TDP-43, with reduced toxicity due to pbp1Δ. However, extragenic modifiers and TDP-43 mutants that reduced TDP-43 toxicity, but not TDP-43 mutants that enhanced toxicity, restored TOROID formation. This is consistent with the hypothesis that TDP-43 is toxic in yeast because it reduces TOROID formation, causing the inhibition of autophagy. Whether TDP-43 exerts a similar effect in higher cells remains to be determined.

Calcium-responsive transactivator (CREST) toxicity is rescued by loss of PBP1/ATXN2 function in a novel yeast proteinopathy model and in transgenic flies.

Proteins associated with familial neurodegenerative disease often aggregate in patients' neurons. Several such proteins, e.g. TDP-43, aggregate and are toxic when expressed in yeast. Deletion of the ATXN2 ortholog, PBP1, reduces yeast TDP-43 toxicity, which led to identification of ATXN2 as an amyotrophic lateral sclerosis (ALS) risk factor and therapeutic target. Likewise, new yeast neurodegenerative disease models could facilitate identification of other risk factors and targets. Mutations in SS18L1, encoding the calcium-responsive transactivator (CREST) chromatin-remodeling protein, are associated with ALS. We show that CREST is toxic in yeast and forms nuclear and occasionally cytoplasmic foci that stain with Thioflavin-T, a dye indicative of amyloid-like protein. Like the yeast chromatin-remodeling factor SWI1, CREST inhibits silencing of FLO genes. Toxicity of CREST is enhanced by the [PIN+] prion and reduced by deletion of the HSP104 chaperone required for the propagation of many yeast prions. Likewise, deletion of PBP1 reduced CREST toxicity and aggregation. In accord with the yeast data, we show that the Drosophila ortholog of human ATXN2, dAtx2, is a potent enhancer of CREST toxicity. Downregulation of dAtx2 in flies overexpressing CREST in retinal ganglion cells was sufficient to largely rescue the severe degenerative phenotype induced by human CREST. Overexpression caused considerable co-localization of CREST and PBP1/ATXN2 in cytoplasmic foci in both yeast and mammalian cells. Thus, co-aggregation of CREST and PBP1/ATXN2 may serve as one of the mechanisms of PBP1/ATXN2-mediated toxicity. These results extend the spectrum of ALS associated proteins whose toxicity is regulated by PBP1/ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases.

Long non-coding RNA NEAT1_1 ameliorates TDP-43 toxicity in in vivo models of TDP-43 proteinopathy.

Pathological changes involving TDP-43 protein ('TDP-43 proteinopathy') are typical for several neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). FTLD-TDP cases are characterized by increased binding of TDP-43 to an abundant lncRNA, NEAT1, in the cortex. However it is unclear whether enhanced TDP-43-NEAT1 interaction represents a protective mechanism. We show that accumulation of human TDP-43 leads to upregulation of the constitutive NEAT1 isoform, NEAT1_1, in cultured cells and in the brains of transgenic mice. Further, we demonstrate that overexpression of NEAT1_1 ameliorates TDP-43 toxicity in Drosophila and yeast models of TDP-43 proteinopathy. Thus, NEAT1_1 upregulation may be protective in TDP-43 proteinopathies affecting the brain. Approaches to boost NEAT1_1 expression in the CNS may prove useful in the treatment of these conditions.

Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43's effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS.

Prion induction by the short-lived, stress-induced protein Lsb2 is regulated by ubiquitination and association with the actin cytoskeleton.

Yeast prions are self-perpetuating, QN-rich amyloids that control heritable traits and serve as a model for mammalian amyloidoses. De novo prion formation by overproduced prion protein is facilitated by other aggregated QN-rich protein(s) and is influenced by alterations of protein homeostasis. Here we explore the mechanism by which the Las17-binding protein Lsb2 (Pin3) promotes conversion of the translation termination factor Sup35 into its prion form, [PSI(+)]. We show that Lsb2 localizes with some Sup35 aggregates and that Lsb2 is a short-lived protein whose levels are controlled via the ubiquitin-proteasome system and are dramatically increased by stress. Loss of Lsb2 decreases stability of [PSI(+)] after brief heat shock. Mutations interfering with Lsb2 ubiquitination increase prion induction, while a mutation eliminating association of Lsb2 with the actin cytoskeleton blocks its aggregation and prion-inducing ability. These findings directly implicate the UPS and actin cytoskeleton in regulating prions via a stress-inducible QN-rich protein.

Heterologous aggregates promote de novo prion appearance via more than one mechanism.

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN+]-independent pathway.

Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller "seeds." We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI(+)] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI(+)] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI(+)] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI(+)].

Prion-promoted phosphorylation of heterologous amyloid is coupled with ubiquitin-proteasome system inhibition and toxicity.

Many neurodegenerative diseases are associated with conversion of a soluble protein into amyloid deposits, but how this is connected to toxicity remains largely unknown. Here, we explore mechanisms of amyloid associated toxicity using yeast. [PIN(+)], the prion form of the Q/N-rich Rnq1 protein, was known to enhance aggregation of heterologous proteins, including the overexpressed Q/N-rich amyloid forming domain of Pin4 (Pin4C), and Pin4C aggregates were known to attract chaperones, including Sis1. Here we show that in [PIN(+)] but not [pin(-)] cells, overexpression of Pin4C is deadly and linked to hyperphosphorylation of aggregated Pin4C. Furthermore, Pin4C aggregation, hyperphosphorylation and toxicity are simultaneously reversed by Sis1 overexpression. Toxicity may result from proteasome overload because hyperphosphorylated Pin4C aggregation is associated with reduced degradation of a ubiquitin-protein degradation reporter. Finally, hyperphosphorylation of endogenous full-length Pin4 was also facilitated by [PIN(+)], revealing that a prion can regulate post-translational modification of another protein.

Prion formation and polyglutamine aggregation are controlled by two classes of genes.

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN+]. When fused to GFP and overexpressed in [ps⁻] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI+] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI+]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI+] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI+] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.

Exploring Fundamentals of Prion Biology Using Natural Yeast Prions and Mammalian PrP.

The key postulate of the prion paradigm is that some proteins can take on unconventional conformations and pass these conformations to newly synthesized protein molecules with the same primary structure [...].

[PSI(+) ] prion variant establishment in yeast.

Differences in the clinical pathology of mammalian prion diseases reflect distinct heritable conformations of aggregated PrP proteins, called prion strains. Here, using the yeast [PSI(+) ] prion, we examine the de novo establishment of prion strains (called variants in yeast). The [PSI(+) ] prion protein, Sup35, is efficiently induced to take on numerous prion variant conformations following transient overexpression of Sup35 in the presence of another prion, e.g. [PIN(+) ]. One hypothesis is that the first [PSI(+) ] prion seed to arise in a cell causes propagation of only that seed's variant, but that different variants could be initiated in different cells. However, we now show that even within a single cell, Sup35 retains the potential to fold into more than one variant type. When individual cells segregating different [PSI(+) ] variants were followed in pedigrees, establishment of a single variant phenotype generally occurred in daughters, granddaughters or great-granddaughters - but in 5% of the pedigrees cells continued to segregate multiple variants indefinitely. The data are consistent with the idea that many newly formed prions go through a maturation phase before they reach a single specific variant conformation. These findings may be relevant to mammalian PrP prion strain establishment and adaptation.

Comment on Billant et al. p53, A Victim of the Prion Fashion. Cancers 2021, 13, 269.

The p53 tumor suppressor is a central protein in the fight against cancer [...].

TDP-43 Toxicity in Yeast Is Associated with a Reduction in Autophagy, and Deletions of TIP41 and PBP1 Counteract These Effects.

When human TDP-43 is overexpressed in yeast it is toxic and forms cytoplasmic aggregates. The mechanism of this toxicity is unknown. Genetic screens for TDP-43 toxicity modifiers in the yeast system previously identified proteins, including PBP1, that enhance TDP-43 toxicity. The determination in yeast that deletion of PBP1 reduces TDP-43 toxicity while overexpression enhances toxicity, led to the discovery that its human homolog, ATXN2, is associated with ALS risk. Thus, the yeast system has relevance to human disease. We now show that deletion of a new yeast gene, tip41Δ, likewise suppresses TDP-43 toxicity. We also found that TDP-43 overexpression and toxicity is associated with reduced autophagy. This is consistent with findings in other systems that increasing autophagy reduces TDP-43 toxicity and is in contrast to a report of enhanced autophagy when TDP-43 was overexpressed in yeast. Interestingly, we found that deletions of PBP1 and TIP41, which reduced TDP-43 toxicity, eliminated TDP-43's inhibition of autophagy. This suggests that toxicity of TDP-43 expressed in yeast is in part due to its inhibition of autophagy and that deletions of PBP1 and TIP41 may reduce TDP-43 toxicity by preventing TDP-43 from inhibiting autophagy.

A new colour assay for [URE3] prion in a genetic background used to score for the [PSI⁺] prion.

Most scoring assays for yeast prions are dependent on specific genetic markers and constructs that differ for each prion. Here we describe a simple colour assay for the [URE3] prion that works in the 74D-964 strain frequently used to score the [PSI(+)] prion. Although this assay can only be used to score for [URE3] in the [psi(-)] version of the strain, it makes it easier to examine the effects of host mutations or environmental changes on [URE3] or [PSI(+)] using a colour assay in the identical genetic background.

Tumor suppressor protein p53 expressed in yeast can remain diffuse, form a prion, or form unstable liquid-like droplets.

Mutations in the p53 tumor suppressor are frequent causes of cancer. Because p53 aggregates appear in some tumor cells, it has been suggested that p53 could also cause cancer by forming self-replicating protein aggregates (prions). Here, using yeast, we show that transient p53 overexpression induced the formation of p53 prion aggregates that were transmitted for >100 generations, found in lysate pellets, stained with Thioflavin T, and transmitted by cytoplasmic transfer, or transfection with lysates of cells carrying the prion or with p53 amyloid peptide. As predicted for a prion, transient interruption of p53 expression caused permanent p53 prion loss. Importantly, p53 transcription factor activity was reduced by prion formation suggesting that prion aggregation could cause cancer. p53 has also been found in liquid-like nuclear droplets in animal cell culture. In yeast, we found that liquid-like p53 foci appear in response to stress and disappear with stress removal.

Sequestration of essential proteins causes prion associated toxicity in yeast.

Prions are infectious, aggregated proteins that cause diseases in mammals but are not normally toxic in fungi. Excess Sup35p, an essential yeast protein that can exist as the [PSI(+)] prion, inhibits growth of [PSI(+)] but not [psi(-)] cells. This toxicity is rescued by expressing the Sup35Cp domain of Sup35p, which is sufficient for cell viability but not prion propagation. We now show that rescue requires Sup35Cp levels to be proportional to Sup35p overexpression. Overexpression of Sup35p appeared to cause pre-existing [PSI(+)] aggregates to coalesce into larger aggregates, but these were not toxic per se because they formed even when Sup35Cp rescued growth. Overexpression of Sup45p, but not other tested essential Sup35p binding partners, caused rescue. Sup45-GFPp formed puncta that colocalized with large [PSI(+)] Sup35-RFPp aggregates in cells overexpressing Sup35p, and the frequency of the Sup45-GFPp puncta was reduced by rescuing levels of Sup35Cp. In contrast, [PSI(+)] toxicity caused by a high excess of the Sup35p prion domain (Sup35NMp) was rescued by a single copy of Sup35Cp, was not rescued by Sup45p overexpression and was not associated with the appearance of Sup45-GFPp puncta. This suggests [PSI(+)] toxicity caused by excess Sup35p verses Sup35NMp is, respectively, through sequestration/inactivation of Sup45p verses Sup35p.

Most, but not all, yeast strains in the deletion library contain the [PIN(+)] prion.

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non-Mendelian element, such as a prion, within this library could affect the outcome of many large-scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN(+)] prion. [PIN(+)] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1-GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN(+)]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN(+)], 139 deletions were able to efficiently maintain three different [PIN(+)] variants despite extensive growth and storage at 4 degrees C. One strain, cue2Delta, displayed an alteration in the RNQ1-GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild-type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN(+)], indicating that 5153 non-essential genes are not required for [PIN(+)] propagation.

Heterologous cross-seeding mimics cross-species prion conversion in a yeast model.

BACKGROUND: Prions are self-perpetuating, infectious, aggregated proteins that are associated with several neurodegenerative diseases in mammals and heritable traits in yeast. Sup35p, the protein determinant of the yeast prion [PSI+], has a conserved C terminal domain that performs the Sup35p function and a prion domain that is highly divergent. Prions formed by chimeras of the prion domain of various species fused to the C domain of Saccharomyces cerevisiae exhibit a 'species barrier', a phenomenon first observed in mammals, and often fail to transmit the prion state to chimeras with prion domains of other species. RESULTS: We focus on the chimera containing the prion domain of Pichia methanolica and examine how tight the 'species barrier' is between the chimera and S. cerevisiae. Although either of two Q/N-rich prions, [PSI+] or [PIN+], enhances the formation of the chimeric prion, [CHI+PM], neither a non-Q/N-rich prion nor a non-prion Q-rich aggregate promotes the formation of [CHI+PM]. [CHI+PM] has many features characteristic of yeast prions: aggregation, cytoplasmic transmission and a two-level protein structure. [CHI+PM] formed in the presence of [PSI+] can propagate independently of [PSI+] and forms at least two different variants of the prion, suggesting the generation and not transmission of new prion seeds. CONCLUSION: Although the sequence similarity between the S. cerevisiae Q/N-rich prion determinants and the P. methanolica prion domain is low, we find that the chimera containing the prion domain of P. methanolica can occasionally be cross-seeded by [PSI+] to mimic crossing the species barrier, to form the [CHI+PM] prion. Our data suggests that crossing the barrier occurs by a de novo formation of the foreign chimeric prion. Thus, the species barrier appears to be crossed by a heterologous seeding mechanism, wherein the infected prion protein uses the pre-existing seed as an inefficient template.

Respiration Enhances TDP-43 Toxicity, but TDP-43 Retains Some Toxicity in the Absence of Respiration.

The trans-activating response DNA-binding protein 43 (TDP-43) is a transcriptional repressor and splicing factor. TDP-43 is normally mostly in the nucleus, although it shuttles to the cytoplasm. Mutations in TDP-43 are one cause of familial amyotrophic lateral sclerosis. In neurons of these patients, TDP-43 forms cytoplasmic aggregates. In addition, wild-type TDP-43 is also frequently found in neuronal cytoplasmic aggregates in patients with neurodegenerative diseases not caused by TDP-43 mutations. TDP-43 expressed in yeast causes toxicity and forms cytoplasmic aggregates. This disease model has been validated because genetic modifiers of TDP-43 toxicity in yeast have led to the discovery that their conserved genes in humans are amyotrophic lateral sclerosis genetic risk factors. While how TDP-43 is associated with toxicity is unknown, several studies find that TDP-43 alters mitochondrial function. We now report that TDP-43 is much more toxic when yeast are respiring than when grown on a carbon source where respiration is inhibited. However, respiration is not the unique target of TDP-43 toxicity because we found that TDP-43 retains some toxicity even in the absence of respiration. We found that H2O2 increases the toxicity of TDP-43, suggesting that the reactive oxygen species associated with respiration could likewise enhance the toxicity of TDP-43. In this case, the TDP-43 toxicity targets in the presence or absence of respiration could be identical, with the reactive oxygen species produced by respiration activating TDP-43 to become more toxic or making TDP-43 targets more vulnerable.