Expression of an expanded polyglutamine domain in yeast causes death with apoptotic markers

Huntington's disease is caused by specific mutations in huntingtin protein. Expansion of a polyglutamine (polyQ) repeat of huntingtin leads to protein aggregation in neurons followed by cell death with apoptotic markers. The connection between the aggregation and the degeneration of neurons is poorly understood. Here, we show that the physiological consequences of expanded polyQ domain expression in yeast are similar to those in neurons. In particular, expression of expanded polyQ in yeast causes apoptotic changes in mitochondria, caspase activation, nuclear DNA fragmentation and death. Similar to neurons, at the late stages of expression the expanded polyQ accumulates in the nuclei and seems to affect the cell cycle of yeast. Interestingly, nuclear localization of the aggregates is dependent on functional caspase Yca1. We speculate that the aggregates in the nuclei disturb the cell cycle and thus contribute to the development of the cell death process in both systems. Our data show that expression of the polyQ construct in yeast can be used to model patho-physiological effects of polyQ expansion in neurons.     

Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.     

Analyzing the aggregation of polyglutamine-expansion proteins and its modulation by molecular chaperones

Polyglutamine (polyQ)-expansion proteins cause protein aggregation in the cytosol and nucleus of neuronal cells, leading to neurodegenerative diseases. For example, expansion of the polyQ tract (>40 repeats) in huntingtin (htt) proteins leads to Huntington disease, while polyQ-expanded ataxins cause several types of ataxias. PolyQ-rich inclusions are found in neuronal cells of patients, suggesting that polyQ disease is caused by protein misfolding. However, the mechanisms by which polyQ-expansion proteins exert neuronal toxicity are largely unknown. Here, we review experimental procedures to analyze the roles of molecular chaperones in preventing polyQ aggregation and toxicity as well as to measure the characteristics and dynamics of polyQ aggregation, particularly focusing on cellular models and dynamic imaging of fluorescently-labeled polyQ-expansion proteins and their modulation by chaperones.     

Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.     

Evolution and function of CAG/polyglutamine repeats in protein-protein interaction networks

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions.     

Structural insights into the aggregation mechanism of huntingtin exon 1 protein fragment with different polyQ-lengths

Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N-terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the β-sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all-atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q₂₇, Q₂₇P₁₁, Q₃₄, Q₃₅, Q₃₆, Q₄₀, Q₅₀, and Q₅₀P₁₁. In the models without flanking polyPs, we noticed that the transformation of a random coil to β-sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a β-sheet structure. When polyQ length increases, the 17 N-terminal flanking residues are more likely to adopt a β-sheet conformation from α-helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.     

Nuclear aggregation of huntingtin is not prevented by deletion of chaperone Hsp104

Polyglutamine expansion causes the disease proteins to aggregate, resulting in stable insoluble aggregates in the nucleus. The in vitro aggregation and cellular toxicity of polyglutamine proteins are reduced by chaperone heat shock proteins (Hsp). In polyglutamine disease animal models, however, polyglutamine inclusions remain in the nucleus despite the suppression of neurodegeneration by Hsp. Studies using yeast genetic approach revealed that the balance of Hsp is important for regulating protein aggregation in the cytoplasm of yeast cells. Here we report that N-terminal fragments of huntingtin with an expanded polyglutamine tract form aggregates only in the cytoplasm of yeast cells and, when tagged with nuclear localization sequences (NLS), are able to aggregate in the nucleus. Deletion of the Hsp104 gene prevents the aggregation of huntingtin in the cytoplasm but is unable to eliminate the aggregation of NLS-tagged huntingtin in the nucleus. The inhibitory effect of Hsp104 deletion on the cytoplasmic aggregation of huntingtin only occurs in viable yeast cells, as aggregates can be formed in Hsp104 deletion cells that have been frozen for 72 h. Fresh cytosolic extracts of the Hsp104 deletion strain inhibit the aggregation of huntingtin in vitro, suggesting that the deletion of Hsp104 may alter the activities of other cytoplasmic factors to inhibit polyglutamine aggregation in the cytoplasm. We propose that the regulatory effects of chaperones may mainly be restricted to the cytoplasm and have much less influence on polyglutamine-containing aggregates in the nucleus.     

A Coarse-Grained Model for Polyglutamine Aggregation Modulated by Amphipathic Flanking Sequences

The aggregation of proteins with expanded polyglutamine (polyQ) tracts is directly relevant to the formation of neuronal intranuclear inclusions in Huntington’s disease. In vitro studies have uncovered the effects of flanking sequences as modulators of the driving forces and mechanisms of polyQ aggregation in sequence segments associated with HD. Specifically, a seventeen-residue amphipathic stretch (N17) that is directly N-terminal to the polyQ tract in huntingtin decreases the overall solubility, destabilizes nonfibrillar aggregates, and accelerates fibril formation. Published results from atomistic simulations showed that the N17 module reduces the frequency of intermolecular association. Our reanalysis of these simulation results demonstrates that the N17 module also reduces interchain entanglements between polyQ domains. These two effects, which are observed on the smallest lengthscales, are incorporated into phenomenological pair potentials and used in coarse-grained Brownian dynamics simulations to investigate their impact on large-scale aggregation. We analyze the results from Brownian dynamics simulations using the framework of diffusion-limited cluster aggregation. When entanglements prevail, which is true in the absence of N17, small spherical clusters and large linear aggregates form on distinct timescales, in accord with in vitro experiments. Conversely, when entanglements are quenched and a barrier to intermolecular associations is introduced, both of which are attributable to N17, the timescales for forming small species and large linear aggregates become similar. Therefore, the combination of a reduction of interchain entanglements through homopolymeric polyQ and barriers to intermolecular associations appears to be sufficient for providing a minimalist phenomenological rationalization of in vitro observations regarding the effects of N17 on polyQ aggregation.     

Polyglutamine expansion mutation yields a pathological epitope linked to nucleation of protein aggregate: determinant of Huntington's disease onset

Polyglutamine (polyQ) expansion mutation causes conformational, neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. These diseases are characterized by the aggregation of misfolded proteins, such as amyloid fibrils, which are toxic to cells. Amyloid fibrils are formed by a nucleated growth polymerization reaction. Unexpectedly, the critical nucleus of polyQ aggregation was found to be a monomer, suggesting that the rate-limiting nucleation process of polyQ aggregation involves the folding of mutated protein monomers. The monoclonal antibody 1C2 selectively recognizes expanded pathogenic and aggregate-prone glutamine repeats in polyQ diseases, including Huntington's disease (HD), as well as binding to polyleucine. We have therefore assayed the in vitro and in vivo aggregation kinetics of these monomeric proteins. We found that the repeat-length-dependent differences in aggregation lag times of variable lengths of polyQ and polyleucine tracts were consistently related to the integration of the length-dependent intensity of anti-1C2 signal on soluble monomers of these proteins. Surprisingly, the correlation between the aggregation lag times of polyQ tracts and the intensity of anti-1C2 signal on soluble monomers of huntingtin precisely reflected the repeat-length dependent age-of-onset of HD patients. These data suggest that the alterations in protein surface structure due to polyQ expansion mutation in soluble monomers of the mutated proteins act as an amyloid-precursor epitope. This, in turn, leads to nucleation, a key process in protein aggregation, thereby determining HD onset. These findings provide new insight into the gain-of-function mechanisms of polyQ diseases, in which polyQ expansion leads to nucleation rather than having toxic effects on the cells.     

The Role of Interruptions in polyQ in the Pathology of SCA1

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.     

A Coarse-Grained Model for Polyglutamine Aggregation Modulated by Amphipathic Flanking Sequences

The aggregation of proteins with expanded polyglutamine (polyQ) tracts is directly relevant to the formation of neuronal intranuclear inclusions in Huntington’s disease. In vitro studies have uncovered the effects of flanking sequences as modulators of the driving forces and mechanisms of polyQ aggregation in sequence segments associated with HD. Specifically, a seventeen-residue amphipathic stretch (N17) that is directly N-terminal to the polyQ tract in huntingtin decreases the overall solubility, destabilizes nonfibrillar aggregates, and accelerates fibril formation. Published results from atomistic simulations showed that the N17 module reduces the frequency of intermolecular association. Our reanalysis of these simulation results demonstrates that the N17 module also reduces interchain entanglements between polyQ domains. These two effects, which are observed on the smallest lengthscales, are incorporated into phenomenological pair potentials and used in coarse-grained Brownian dynamics simulations to investigate their impact on large-scale aggregation. We analyze the results from Brownian dynamics simulations using the framework of diffusion-limited cluster aggregation. When entanglements prevail, which is true in the absence of N17, small spherical clusters and large linear aggregates form on distinct timescales, in accord with in vitro experiments. Conversely, when entanglements are quenched and a barrier to intermolecular associations is introduced, both of which are attributable to N17, the timescales for forming small species and large linear aggregates become similar. Therefore, the combination of a reduction of interchain entanglements through homopolymeric polyQ and barriers to intermolecular associations appears to be sufficient for providing a minimalist phenomenological rationalization of in vitro observations regarding the effects of N17 on polyQ aggregation.     

Wild type huntingtin toxicity in yeast: Implications for the role of amyloid cross-seeding in polyQ diseases

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.     

Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis

The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.     

Evidence for a recruitment and sequestration mechanism in Huntington's disease.

Polyglutamine (polyQ) extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyQ aggregates in Huntington's disease. We constructed an array of CAG/CAA triplet repeats, coding for a range of 25-300 glutamine residues, which was used to generate expression constructs with minimal flanking sequence. Normal-length (25 glutamine residues) polyQ did not aggregate when transfected alone. Remarkably, when co-transfected with extended (100-300 glutamine residues) polyQ tracts, normal-length polyQ-containing peptides were trapped in insoluble detergent-resistant aggregates. Aggregates formed in the cytoplasm but were visible in the nucleus only when a strong nuclear localization signal was present. Intermolecular interactions between polyQ tracts mediated the localization of heterogeneous aggregates into the nucleolus by nucleolin protein. Our results suggest that extended polyQ can interact with cellular polyQ-containing proteins, transport them to ectopic cellular locations, and form heterogeneous polyQ aggregates. We provide evidence for a recruitment mechanism for pathogenesis in the polyQ neurodegenerative disorders. In susceptible cells, extended polyQ tracts in huntingtin might interact with and sequester or deplete certain endogenous polyQ-containing cellular proteins.     

Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins

Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.     

The Machado–Joseph disease‐associated form of ataxin‐3 impacts dynamics of clathrin‐coated pits

Expansion above a certain threshold in the polyglutamine (polyQ) tract of ataxin‐3 is the main cause of neurodegeneration in Machado–Joseph disease. Ataxin‐3 contains an N‐terminal catalytic domain, called Josephin domain, and a highly aggregation‐prone C‐terminal domain containing the polyQ tract. Recent work has shown that protein aggregation inhibits clathrin‐mediated endocytosis (CME). However, the effects of polyQ expansion in ataxin‐3 on CME have not been investigated. We hypothesize that the expansion of the polyQ tract in ataxin‐3 could impact CME. Here, we report that both the wild‐type and the expanded ataxin‐3 reduce transferrin internalization and expanded ataxin‐3 impacts dynamics of clathrin‐coated pits (CCPs) by reducing CCP nucleation and increasing short‐lived abortive CCPs. Since endocytosis plays a central role in regulating receptor uptake and cargo release, our work highlights a potential mechanism linking protein aggregation to cellular dysregulation.     

Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity

The polyglutamine (polyQ) diseases are a class of inherited neurodegenerative diseases including Huntington's disease, caused by the expansion of a polyQ stretch within each disease protein. This expansion is thought to cause a conformational change in the protein leading to aggregation of the protein, resulting in cytotoxicity. To analyze whether disrupting the toxic conformation of the polyQ protein can alter its aggregation propensity and cytotoxicity, we examined the effect of interruption of the expanded polyQ stretch by proline insertion, since prolines cause great alterations in protein conformation. Here, we show that insertion of prolines into the expanded polyQ stretch indeed disrupts its ordered secondary structure, leading to suppression of polyQ protein aggregation both in vitro and in cell culture, and reduction of cytotoxicity in correlation with the number of proline interruptions. Furthermore, we found that a short polyQ stretch with a proline interruption is able to inhibit aggregation of the expanded polyQ protein in trans. These results show that a gain in toxic conformation of the expanded polyQ protein is essential for aggregation and cytotoxicity, providing insight into establishing therapies against the polyQ diseases.