Background: Yeast have been used to study hungtingtin toxicity.
Results: Both HttQ103 and HttQP103 are toxic in yeast with [PSI+] prion. This toxicity is markedly rescued by a Sup35 fragment.
Conclusion: Sequestration of the essential protein, Sup35, contributes to Htt toxicity in yeast.
Significance: This research demonstrates the complex nature of Htt toxicity.
Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN+] prion, which is the amyloid form of Rnq1, or [PSI+] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI+] yeast than in [PIN+], even though HttQP103 formed multiple aggregates in both [PSI+] and [PIN+] yeast. This toxicity was only observed in the strong [PSI+] variant, not the weak [PSI+] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI+] prion is primarily due to sequestration of the essential protein, Sup35.
Huntington Disease; Prions; Protein Folding; Yeast; Yeast Physiology
Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (HTT) protein. The expression of mutant HTT in the baker’s yeast Saccharomyces cerevisiae recapitulates many of the cellular phenotypes observed in mammalian HD models. Mutant HTT aggregation and toxicity in yeast is influenced by the presence of the Rnq1p and Sup35p prions, as well as other glutamine/asparagine-rich aggregation-prone proteins. Here we investigated the ability of a subset of these proteins to modulate mutant HTT aggregation and to substitute for the prion form of Rnq1p. We find that overexpression of either the putative prion Ybr016wp or the Sup35p prion restores aggregation of mutant HTT in yeast cells lacking the Rnq1p prion. These results indicate that an interchangeable suite of aggregation-prone proteins regulates mutant HTT aggregation dynamics in yeast, which may have implications for mutant HTT aggregation in human cells.
Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.
Polyglutamine diseases, including Huntington disease, are associated with expansions of polyglutamine tracts, resulting in aggregation of respective proteins. The severity of Huntington disease is controlled by both DNA and non–DNA factors. Mechanisms of such a control are poorly understood. Polyglutamine may sequester other cellular proteins; however, different experimental models have pointed to different sequestered proteins. By using a yeast model, we demonstrate that the mechanism of polyglutamine toxicity is driven by the composition of other (endogenous) aggregates (for example, yeast prions) present in a eukaryotic cell. Although these aggregates do not necessarily cause significant toxicity on their own, they serve as mediators in protein sequestration and therefore determine which specific proteins are to be sequestered by polyglutamines. We also show that polyglutamine deposition into an aggresome, a perinuclear compartment thought to be cytoprotective, fails to ameliorate cytotoxicity in cells with certain compositions of pre-existing aggregates. Finally, we demonstrate that an increase in the dosage of a sequestered protein due to aneuploidy by a chromosome carrying a respective gene may rescue cytotoxicity. Our data shed light on genetic and epigenetic mechanisms modulating polyglutamine cytotoxicity and establish a new approach for identifying potential therapeutic targets through characterization of the endogenous aggregated proteins.
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.
Certain proteins can misfold into β-sheet-rich, self-seeding aggregates. Such proteins appear to be associated with neurodegenerative diseases such as prion, Alzheimer's and Parkinson's. Yeast prions also misfold into self-seeding aggregates and provide a good model to study how these rogue polymers first appear. De novo prion appearance can be made very frequent in yeast by transient overexpression of the prion protein in the presence of heterologous prions or prion-like aggregates. Here, we show that the aggregates of one such newly induced prion are initially formed in a dot-like structure near the vacuole. These dots then grow into rings at the periphery of the cell prior to becoming smaller rings surrounding the vacuole and maturing into the characteristic heritable prion tiny dots found throughout the cytoplasm. We found considerable colocalization of two heterologous prion/prion-like aggregates with the newly appearing prion protein aggregates, which is consistent with the prevalent model that existing prion aggregates can cross-seed the de novo aggregation of a heterologous prion protein. However, we failed to find any physical interaction between another heterologous aggregating protein and the newly appearing prion aggregates it stimulated to appear, which is inconsistent with cross-seeding.
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.
Certain proteins that exist in functional unaggregated conformers can also form self-perpetuating infectious aggregates called prions. Here we investigate factors involved in the initial switch to the prion form. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by overexpression of the SUP35 gene in the presence of the prion form of the Rnq1 protein, [PIN+]. When tagged with green fluorescent protein and transiently overexpressed in [psi−] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings give rise to daughter cells that are [PSI+]. Here, we investigate factors required for this induction of [PSI+]. Analyses of over 400 gene deletions revealed two classes that reduce [PSI+] induction: one class forms fluorescent rings normally, and the other does not. Interestingly, the former class enhanced, while the latter class reduced, toxicity associated with the expanded polyglutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. These results suggest that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.
The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, β-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (≥54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.
Expansion of a stretch of polyglutamine in huntingtin (htt), the protein product of the IT15 gene, causes Huntington's disease (HD). Previous investigations into the role of the polyglutamine stretch (polyQ) in htt function have suggested that its length may modulate a normal htt function involved in regulating energy homeostasis. Here we show that expression of full-length htt lacking its polyglutamine stretch (ΔQ-htt) in a knockin mouse model for HD (Hdh140Q/ΔQ), reduces significantly neuropil mutant htt aggregates, ameliorates motor/behavioral deficits, and extends lifespan in comparison to the HD model mice (Hdh140Q/+). The rescue of HD model phenotypes is accompanied by the normalization of lipofuscin levels in the brain and an increase in the steady-state levels of the mammalian autophagy marker microtubule-associate protein 1 light chain 3-II (LC3-II). We also find that ΔQ-htt expression in vitro increases autophagosome synthesis and stimulates the Atg5-dependent clearance of truncated N-terminal htt aggregates. ΔQ-htt's effect on autophagy most likely represents a gain-of-function, as overexpression of full-length wild-type htt in vitro does not increase autophagosome synthesis. Moreover, HdhΔQ/ΔQ mice live significantly longer than wild-type mice, suggesting that autophagy upregulation may be beneficial both in diseases caused by toxic intracellular aggregate-prone proteins and also as a lifespan extender in normal mammals.
Expansion of a stretch of glutamines near the amino-terminus of huntingtin (htt), the protein product of the IT15 gene, is a deleterious mutation that causes Huntington's disease (HD). Here we show, in contrast, that deletion of htt's normal polyglutamine stretch (ΔQ-htt) is a potentially beneficial mutation that can ameliorate HD mouse model phenotypes when ΔQ-htt is expressed together with a version of htt with the HD mutation. In addition, ΔQ-htt expression can enhance longevity when expressed in either an HD mouse model or in non–HD mice. ΔQ-htt's effects on both lifespan and HD model phenotypes are likely due to an increase in autophagy, a major recycling pathway in cells that is involved in the turnover of cellular components, and aggregated protein. Based on our results, we suggest that development of therapeutic agents that can stimulate autophagy may help both in treating neurodegenerative disorders like HD and also in increasing longevity.
Escape of aberrant proteins from protein quality control leads to accumulation of toxic protein species. Sti1 interacts with Hsp70 to mediate spatial PQC of amyloid-like proteins by regulating their distribution in different intracellular protein-handling depots. Sti1 suppresses proteotoxicity by targeting amyloid-like proteins to perinuclear foci.
Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1–monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.
Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q) toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS). High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic aggregation intermediates to the Hsp70/Sti1 system for spatial organization into benign species.
In yeast, fragmentation of amyloid polymers by the Hsp104 chaperone allows them to propagate as prions. The prion-forming domain of the yeast Sup35 protein is rich in glutamine, asparagine, tyrosine, and glycine residues, which may define its prion properties. Long polyglutamine stretches can also drive amyloid polymerization in yeast, but these polymers are unable to propagate because of poor fragmentation and exist through constant seeding with the Rnq1 prion polymers. We proposed that fragmentation of polyglutamine amyloids may be improved by incorporation of hydrophobic amino acid residues into polyglutamine stretches. To investigate this, we constructed sets of polyglutamine with or without tyrosine stretches fused to the non-prion domains of Sup35. Polymerization of these chimeras started rapidly, and its efficiency increased with stretch size. Polymerization of proteins with polyglutamine stretches shorter than 70 residues required Rnq1 prion seeds. Proteins with longer stretches polymerized independently of Rnq1 and thus could propagate. The presence of tyrosines within polyglutamine stretches dramatically enhanced polymer fragmentation and allowed polymer propagation in the absence of Rnq1 and, in some cases, of Hsp104.
Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%–4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.
Huntington's Disease (HD) is a fatal inherited neurodegenerative disease, which typically begins in middle age and progresses with symptoms of severe uncontrolled movements and cognitive dysfunction. HD is uniformly fatal with death occurring ten to 15 years after onset of symptoms. There is currently no effective treatment for HD. The genetic mutation underlying HD causes a protein called huntingtin (Htt) to contain an abnormally long tract of the amino acid glutamine. This extended span of glutamines changes the shape of the Htt protein, which can cause it to interact in abnormal ways with other cellular proteins. In this study, we have identified a large number of new proteins that bind to normal and mutant forms of the Htt protein. To establish a potential role for these interacting proteins in HD, we show that changing the expression of many of these proteins can modulate the pathological effects of mutant Htt on fly neurons that deteriorate when they express mutant Htt. Identifying cellular proteins that bind to Htt and modulate its pathological activity may facilitate the discovery of an effective treatment for HD.
Huntington's disease (HD) is caused by a polyglutamine expansion within the huntingtin (Htt) protein. Both loss of function of normal Htt and gain of a toxic function by the polyglutamine-expanded mutant Htt protein have been proposed to be responsible for HD, although the molecular mechanisms involved are unclear. We show that Htt is a neuroprotective protein in both HD-related and unrelated model systems. Neuroprotection by Htt is mediated by its sequestration of histone deacetylase-3 (HDAC3), a protein known to promote neuronal death. In contrast to the normal Htt, mutant Htt interacts poorly with HDAC3. However, expression of mutant Htt liberates HDAC3 from Htt, thus de-repressing its neurotoxic activity. Indeed, mutant Htt neurotoxicity is inhibited by the knockdown of HDAC3 and markedly reduced in HDAC3-deficient neurons. A reduction in Htt–HDAC3 interaction is also seen in neurons exposed to other apoptotic stimuli and in the striatum of R6/2 HD mice. Our results suggest that the robust interaction between Htt and HDAC3 along with the ability of mutant Htt to disrupt this association while not itself interacting with HDAC3 provides an explanation for both the loss-of-function and gain-of-toxic-function mechanisms proposed for HD. Moreover, our results identify HDAC3 as an essential player in mutant Htt-induced neurodegeneration.
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ) stretch within Huntingtin (htt), the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR) in modulating HD mouse model pathogenesis is currently unknown.
We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh) encoding N-terminal hemaglutinin (HA) or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt) and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt). Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q) htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis.
In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.
Huntingtin; Epitope tag; Knock-in; Polyglutamine; Proline-rich region; Sequestration; Huntington’s disease
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+].
Certain proteins can occasionally misfold into infectious aggregates called prions. Once formed, these aggregates grow by attracting the soluble form of that protein to join them. The presence of these aggregates can cause profound effects on cells and, in humans, can cause diseases such as transmissible spongiform encephalopathies (TSEs). In yeast, the aggregates are efficiently transmitted to daughter cells because they are cut into small pieces by molecular scissors (chaperones). Here we show that heritable prion aggregates are frequently lost when we overproduce certain other proteins with curing activity. We analyzed one such protein in detail and found that when it is overproduced it forms aggregates that sequester chaperones. This sequestration appears to block the ability of the chaperones to cut the prion aggregates. The result is that the prions get too large to be transmitted to daughter cells. Such sequestration of molecular scissors provides a potential approach to thwart the propagation of disease-causing infectious protein aggregates.
Onset of proteotoxicity is linked to change in the subcellular location of proteins that cause misfolding diseases. Yet, factors that drive changes in disease protein localization and the impact of residence in new surroundings on proteotoxicity are not entirely clear. To address these issues, we examined aspects of proteotoxicity caused by Rnq1-green fluorescent protein (GFP) and a huntingtin's protein exon-1 fragment with an expanded polyglutamine tract (Htt-103Q), which is dependent upon the intracellular presence of [RNQ+] prions. Increasing heat-shock protein 40 chaperone activity before Rnq1-GFP expression, shifted Rnq1-GFP aggregation from the cytosol to the nucleus. Assembly of Rnq1-GFP into benign amyloid-like aggregates was more efficient in the nucleus than cytosol and nuclear accumulation of Rnq1-GFP correlated with reduced toxicity. [RNQ+] prions were found to form stable complexes with Htt-103Q, and nuclear Rnq1-GFP aggregates were capable of sequestering Htt-103Q in the nucleus. On accumulation in the nucleus, conversion of Htt-103Q into SDS-resistant aggregates was dramatically reduced and Htt-103Q toxicity was exacerbated. Alterations in activity of molecular chaperones, the localization of intracellular interaction partners, or both can impact the cellular location of disease proteins. This, in turn, impacts proteotoxicity because the assembly of proteins to a benign state occurs with different efficiencies in the cytosol and nucleus.
Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients, animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression. An understanding of the effects of mHtt may lead to strategies to slow the progression of HD. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of the current models using two N-mHtt-affected promoters in two cell models of HD using overexpression of known N-mHtt-interacting transcription factors, promoter deletion and mutation analyses and in vitro promoter binding assays. Consequently, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at promoters. In this model, N-mHtt interacts with multiple partners whose presence and affinity for N-mHtt influence the severity of gene dysregulation. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.
A number of mouse models expressing mutant huntingtin (htt) with an expanded polyglutamine (polyQ) domain are useful for studying the pathogenesis of Huntington's disease (HD) and identifying appropriate therapies. However, these models exhibit neurological phenotypes that differ in their severity and nature. Understanding how transgenic htt leads to variable neuropathology in animal models would shed light on the pathogenesis of HD and help us to choose HD models for investigation. By comparing the expression of mutant htt at the transcriptional and protein levels in transgenic mice expressing N-terminal or full-length mutant htt, we found that the accumulation and aggregation of mutant htt in the brain is determined by htt context. HD mouse models demonstrating more severe phenotypes show earlier accumulation of N-terminal mutant htt fragments, which leads to the formation of htt aggregates that are primarily present in neuronal nuclei and processes, as well as glial cells. Similarly, transgenic monkeys expressing exon-1 htt with a 147-glutamine repeat (147Q) died early and showed abundant neuropil aggregates in swelling neuronal processes. Fractionation of HD150Q knock-in mice brains revealed an age-dependent accumulation of N-terminal mutant htt fragments in the nucleus and synaptosomes, and this accumulation was most pronounced in the striatum due to decreased proteasomal activity. Our findings suggest that the neuropathological phenotypes of HD stem largely from the accumulation of N-terminal mutant htt fragments and that this accumulation is determined by htt context and cell-type-dependent clearance of mutant htt.
Prions are infectious, self-propagating protein conformations. Rnq1 is required for the yeast Saccharomyces cerevisiae prion [PIN+], which is necessary for the de novo induction of a second prion, [PSI+]. Here we isolated a [PSI+]-eliminating mutant, Rnq1Δ100, that deletes the nonprion domain of Rnq1. Rnq1Δ100 inhibits not only [PSI+] prion propagation but also [URE3] prion and huntingtin's polyglutamine aggregate propagation in a [PIN+] background but not in a [pin−] background. Rnq1Δ100, however, does not eliminate [PIN+]. These findings are interpreted as showing a possible involvement of the Rnq1 prion in the maintenance of heterologous prions and polyQ aggregates. Rnq1 and Rnq1Δ100 form a sodium dodecyl sulfate-stable and Sis1 (an Hsp40 chaperone protein)-containing coaggregate in [PIN+] cells. Importantly, Rnq1Δ100 is highly QN-rich and prone to self-aggregate or coaggregate with Rnq1 when coexpressed in [pin−] cells. However, the [pin−] Rnq1-Rnq1Δ100 coaggregate does not represent a prion-like aggregate. These findings suggest that [PIN+] Rnq1-Rnq1Δ100 aggregates interact with other transmissible and nontransmissible amyloids to destabilize them and that the nonprion domain of Rnq1 plays a crucial role in self-regulation of the highly reactive QN-rich prion domain of Rnq1.
Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI+], [URE3] and [PIN+] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.
prion; Sup35p; Ure2p; Rnq1p; [PSI+]; [URE3]; [PIN+]; amyloid fibrils
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 co-localized 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.
Huntington’s disease (HD) is caused by a mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats at the N-terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knockout mouse (Casp6−/−) Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6−/− mice, regardless of HD genotype. We predicted the Casp6−/− mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6−/− mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6−/− mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 amino acid Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.
Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.
Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal CAG expansion in the Huntingtin gene (HTT), resulting in an expanded polyglutamine track in the HTT protein. Longer CAG expansions correlate with an earlier more severe manifestation of the disease that produces choreic movement, behavioural and psychiatric disturbances, and dementia. Although the causative gene is widely expressed, neuropathology is characterized by striatal and cortical atrophy. HTT interacts with proteins involved in transcription, cell signaling, and transport. The pathogenic role of mutant HTT is not fully understood. This study shows that CAG expanded HTT RNA also contributes to neuronal toxicity. Mutant HTT RNA gives rise to small CAG-repeated RNAs (sCAGs) with neurotoxic activity. These short RNAs interfere with cell functions by silencing the expression of genes that are fully or partially complementary, through a mechanism similar to that of microRNAs. These findings suggest that a small RNA–dependent mechanism may contribute to HD neuronal cell loss. The exhaustive identification of the target genes modulated by sCAGs may lead to a better understanding of HD pathology, allowing the development of new therapeutic strategies.
Aberrant expansion of the number of polyglutamine (polyQ) repeats in mutant proteins is the hallmark of various diseases. These pathologies include Huntington’s disease (HD), a neurological disorder caused by expanded polyQ stretch within the huntingtin (Htt) protein. The expansions increase the propensity of the Htt protein to oligomerize. In the cytoplasm of living cells, the mutant form of Htt (mHtt) is present as soluble monomers and oligomers as well as insoluble aggregates termed inclusion bodies (IBs). Detecting and assessing the relative toxicity of these various forms of mHtt has proven difficult. To enable direct visualization of mHtt soluble oligomers in living cells, we established a split superfolder green fluorescent protein (sfGFP) complementation assay. In this assay, exon 1 variants of Htt (Httex1) containing non-pathological or HD-associated polyQ lengths were fused to two different nonfluorescent fragments of sfGFP. If the Htt proteins oligomerize and the sfGFP fragments come into close proximity, they can associate and complement each other to form a complete and fluorescent sfGFP reporter. Importantly, the irreversible nature of the split-sfGFP complementation allowed us to trap otherwise transient interactions and artificially increase mHtt oligomerization. When coupled with a fluorescent apoptosis reporter, this assay can correlate soluble mHtt oligomer levels and cell death leading to a better characterization of the toxic potential of various forms of mHtt in living cells.
Split-GFP; Huntingtin exon 1; Oligomers; Cell death
Huntington disease (HD) is an inherited neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the huntingtin (Htt) gene. Despite years of research, there is no treatment that extends life for patients with the disorder. Similarly, little is known about which cellular pathways that are altered by pathogenic Huntingtin (Htt) protein expression are correlated with neuronal loss. As part of a longstanding effort to gain insights into HD pathology, we have been studying the protein in the context of the fruitfly Drosophila melanogaster. We generated transgenic HD models in Drosophila by engineering flies that carry a 12-exon fragment of the human Htt gene with or without the toxic trinucleotide repeat expansion. We also created variants with a monomeric red fluorescent protein (mRFP) tag fused to Htt that allows in vivo imaging of Htt protein localization and aggregation. While wild-type Htt remains diffuse throughout the cytoplasm of cells, pathogenic Htt forms insoluble aggregates that accumulate in neuronal soma and axons. Aggregates can physically block transport of numerous organelles along the axon. We have also observed that aggregates are formed quickly, within just a few hours of mutant Htt expression. To explore mechanisms of neurodegeneration in our HD model, we performed in vivo and in vitro screens to search for modifiers of viability and pathogenic Htt aggregation. Our results identified several novel candidates for HD therapeutics that can now be tested in mammalian models of HD. Furthermore, these experiments have highlighted the complex relationship between aggregates and toxicity that exists in HD.
Huntington disease; polyglutamine; Drosophila; aggregates; axonal transport
Neural stem (NS) cells are a limitless resource, and thus superior to primary neurons for drug discovery provided they exhibit appropriate disease phenotypes. Here we established NS cells for cellular studies of Huntington’s disease (HD). HD is a heritable neurodegenerative disease caused by a mutation resulting in an increased number of glutamines (Q) within a polyglutamine tract in Huntingtin (Htt). NS cells were isolated from embryonic wild-type (Htt7Q/7Q) and “knock-in” HD (Htt140Q/140Q) mice expressing full-length endogenous normal or mutant Htt. NS cells were also developed from mouse embryonic stem cells that were devoid of Htt (Htt−/−), or knock-in cells containing human exon1 with an N-terminal FLAG epitope tag and with 7Q or 140Q inserted into one of the mouse alleles (HttF7Q/7Q and HttF140Q/7Q). Compared to Htt7Q/7Q NS cells, HD Htt140Q/140Q NS cells showed significantly reduced levels of cholesterol, increased levels of reactive oxygen species (ROS), and impaired motility. The heterozygous HttF140Q/7Q NS cells had increased ROS and decreased motility compared to HttF7Q/7Q. These phenotypes of HD NS cells replicate those seen in HD patients or in primary cell or in vivo models of HD. Huntingtin “knock-out” NS cells (Htt−/−) also had impaired motility, but in contrast to HD cells had increased cholesterol. In addition, Htt140Q/140Q NS cells had higher phospho-AKT/AKT ratios than Htt7Q/7Q NS cells in resting conditions and after BDNF stimulation, suggesting mutant htt affects AKT dependent growth factor signaling. Upon differentiation, the Htt7Q/7Q and Htt140Q/140Q generated numerous BetaIII-Tubulin- and GABA-positive neurons; however, after 15 days the cellular architecture of the differentiated Htt140Q/140Q cultures changed compared to Htt7Q/7Q cultures and included a marked increase of GFAP-positive cells. Our findings suggest that NS cells expressing endogenous mutant Htt will be useful for study of mechanisms of HD and drug discovery.
embryonic stem cell; mutant huntingtin; neural stem; cholesterol; reactive oxygen species (ROS); reactive oxygen intermediates (ROI); motility; actin; GFAP; astrocytes