Rapid aggregate formation of the huntingtin N-terminal fragment carrying an expanded polyglutamine tract

Huntington's disease (HD) is caused by an expansion of the CAG repeat in the HD gene. The repeat is translated to the polyglutamine tract as huntingtin, the product of HD gene. Several studies showed that the expansion of polyglutamine tract leads to formation of cytoplasminc and/or intranuclear aggregates in vivo or in vitro. To understand the molecular mechanism of the aggregate formation, we studied the transient expression of HD exon 1-GFP fusion proteins in COS-7 cells. The fusion protein carrying 77 glutamine repeats aggregated in a time-dependent manner, while the fusion protein carrying 25 glutamine tract remained to be distributed diffusely in the cytoplasm even 72 hours after transfection. Initially, fluorescent signals were diffusely distributed in the COS-7 cells that were transfected with the construct containing the 77 CAG repeats. Approximately 40 hours later after the transfection, large aggregates grew very rapidly in those cells and the diffuse cytoplasmic fluorescence faded out. This process was completed within 40 minutes from the appearance of small aggregates in the perinuclear regions. The addition of cycloheximide reduced the frequencies of aggregate formation. A possibility was discussed that the aggregate formation was via nucleation. The focal concentration of mutated proteins in neurons may trigger the aggregate formation.     

The Influence of Huntingtin Protein Size on Nuclear Localization and Cellular Toxicity

Huntington disease is an autosomal dominant neurodegenerative disorder caused by the pathological expansion of a polyglutamine tract. In this study we directly assess the influence of protein size on the formation and subcellular localization of huntingtin aggregates. We have created numerous deletion constructs expressing successively smaller fragments of huntingtin and show that these smaller proteins containing 128 glutamines form both intranuclear and perinuclear aggregates. In contrast, larger NH₂-terminal fragments of huntingtin proteins with 128 glutamines form exclusively perinuclear aggregates. These aggregates can form in the absence of endogenous huntingtin. Furthermore, expression of mutant huntingtin results in increased susceptibility to apoptotic stress that is greater with decreasing protein length and increasing polyglutamine size. As both intranuclear and perinuclear aggregates are clearly associated with increased cellular toxicity, this supports an important role for toxic polyglutamine-containing fragments forming aggregates and playing a key role in the pathogenesis of Huntington disease.     

Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the NH(2)-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated NH(2)-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates.     

Circumvention of chaperone requirement for aggregate formation of a short polyglutamine tract by the co-expression of a long polyglutamine tract

Polyglutamine disease is now recognized as one of the conformational, amyloid-related diseases. In this disease, polyglutamine expansion in proteins has toxic effects on cells and also results in the formation of aggregates. Polyglutamine aggregate formation is accompanied by conversion of the polyglutamine from a soluble to an insoluble form. In yeast, the efficiency of the aggregate formation is determined by the balance of various parameters, including the length of the polyglutamine tract, the function of Hsp104, and the level of polyglutamine expression. In this study, we found that the co-expression of a long polyglutamine tract, which formed aggregates independently of the function of Hsp104, enhanced the formation of aggregates of a short polyglutamine tract in wild-type cells as well as in Deltahsp104 mutant cells. Thus, the expression of a long polyglutamine tract would be an additional parameter determining the efficiency of aggregate formation of a short polyglutamine tract. The co-localization of aggregates of long and short polyglutamine tracts suggests the possibility that the enhancement occurs due to the seeding of aggregates of the long polyglutamine tracts.     

Pathogenesis of inclusion bodies in (CAG)n/Qn-expansion diseases with special reference to the role of tissue transglutaminase and to selective vulnerability

At least eight neurodegenerative diseases, including Huntington disease, are caused by expansions in (CAG)n repeats in the affected gene and by an increase in the size of the corresponding polyglutamine domain in the expressed protein. A hallmark of several of these diseases is the presence of aberrant, proteinaceous aggregates in the nuclei and cytosol of affected neurons. Recent studies have shown that expanded polyglutamine (Qn) repeats are excellent glutaminyl-donor substrates of tissue transglutaminase, and that the substrate activity increases with increasing size of the polyglutamine domain. Tissue transglutaminase is present in the cytosol and nuclear fractions of brain tissue. Thus, the nuclear and cytosolic inclusions in Huntington disease may contain tissue transglutaminase-catalyzed covalent aggregates. The (CAG)n/Qn-expansion diseases are classic examples of selective vulnerability in the nervous system, in which certain cells/structures are particularly susceptible to toxic insults. Quantitative differences in the distribution of the brain transglutaminase(s) and its substrates, and in the activation mechanism of the brain transglutaminase(s), may explain in part selective vulnerability in a subset of neurons in (CAG)n-expansion diseases, and possibly in other neurodegenerative disease. If tissue transglutaminase is found to be essential for development of pathogenesis, then inhibitors of this enzyme may be of therapeutic benefit.     

Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat–containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.     

RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells

Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.     

Ubiquitin ligase Hrd1 enhances the degradation and suppresses the toxicity of polyglutamine-expanded huntingtin

E3 ubiquitin ligases catalyze the conjugation of ubiquitin onto proteins, which acts as a signal for targeting proteins for degradation by the proteasome. Hrd1 is an endoplasmic reticulum (ER) membrane-spanning E3 with its catalytic active RING finger facing the cytosol. We speculated that this topology might allow Hrd1 to ubiquitinate misfolded proteins in the cytosol. We tested this idea by using polyglutamine (polyQ)-containing huntingtin (htt) protein as a model substrate. We found that the protein levels of Hrd1 were increased in cells overexpressing the N-terminal fragment of htt containig an expanded polyQ tract (httN). Forced expression of Hrd1 enhanced the degradation of httN in a RING finger-dependent manner, whereas silencing of endogenous Hrd1 expression by RNA interference stabilized httN. Degradation of httN was found to be p97/VCP-dependent, but independent of Ufd1 and Npl4, all of which are thought to form a complex with Hrd1 during ER-associated degradation. Consistent with its role as an E3 for httN, we demonstrate that Hrd1 interacts with and ubiquitinates httN. Subcellular fractionation and confocal microscopy revealed that Hrd1recruits HttN to the ER and co-localizes with juxtanuclear aggregates of httN in cells. Interaction of Hrd1 with httN was found to be independent of the length of the polyglutamine tract. However, httN with expanded polyglutamine tracts appeared to be a preferred substrate for Hrd1. Functionally, we found that Hrd1 protects cells against the httN-induced cell death. These results suggest that Hrd1 is a novel htt-interacting protein that can target pathogenic httN for degradation and is able to protect cells against httN-induced cell death.     

Hsp105alpha suppresses the aggregation of truncated androgen receptor with expanded CAG repeats and cell toxicity

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The N-terminal fragment of AR containing the expanded polyglutamine tract aggregates in cytoplasm and/or in nucleus and induces cell death. Some chaperones such as Hsp40 and Hsp70 have been identified as important regulators of polyglutamine aggregation and/or cell death in neuronal cells. Recently, Hsp105alpha, expressed at especially high levels in mammalian brain, has been shown to suppress apoptosis in neuronal cells and prevent the aggregation of protein caused by heat shock in vitro. However, its role in polyglutamine-mediated cell death and toxicity has not been studied. In the present study, we examined the effects of Hsp105alpha on the aggregation and cell toxicity caused by expansion of the polyglutamine tract using a cellular model of SBMA. The transient expression of truncated ARs (tARs) containing an expanded polyglutamine tract caused aggregates to form in COS-7 and SK-N-SH cells and concomitantly apoptosis in the cells with the nuclear aggregates. When Hsp105alpha was overexpressed with tAR97 in the cells, Hsp105alpha was colocalized to aggregates of tAR97, and the aggregation and cell toxicity caused by expansion of the polyglutamine tract were markedly reduced. Both beta-sheet and alpha-helix domains, but not the ATPase domain, of Hsp105alpha were necessary to suppress the formation of aggregates in vivo and in vitro. Furthermore, Hsp105alpha was found to localize in nuclear inclusions formed by ARs containing an expanded polyglutamine tract in tissues of patients and transgenic mice with SBMA. These findings suggest that overexpression of Hsp105alpha suppresses cell death caused by expansion of the polyglutamine tract without chaperone activity, and the enhanced expression of the essential domains of Hsp105alpha in brain may provide an effective therapeutic approach for CAG repeat diseases.     

Na+/H+ Exchangers Induce Autophagy in Neurons and Inhibit Polyglutamine-Induced Aggregate Formation

In polyglutamine diseases, an abnormally elongated polyglutamine results in protein misfolding and accumulation of intracellular aggregates. Autophagy is a major cellular degradative pathway responsible for eliminating unnecessary proteins, including polyglutamine aggregates. Basal autophagy constitutively occurs at low levels in cells for the performance of homeostatic function, but the regulatory mechanism for basal autophagy remains elusive. Here we show that the Na⁺/H⁺ exchanger (NHE) family of ion transporters affect autophagy in a neuron-like cell line (Neuro-2a cells). We showed that expression of NHE1 and NHE5 is correlated to polyglutamine accumulation levels in a cellular model of Huntington''s disease, a fatal neurodegenerative disorder characterized by accumulation of polyglutamine-containing aggregate formation in the brain. Furthermore, we showed that loss of NHE5 results in increased polyglutamine accumulation in an animal model of Huntington''s disease. Our data suggest that cellular pH regulation by NHE1 and NHE5 plays a role in regulating basal autophagy and thereby promotes autophagy-mediated degradation of proteins including polyglutamine aggregates.     

Structural characterization of transglutaminase-catalyzed cross-linking between glyceraldehyde 3-phosphate dehydrogenase and polyglutamine repeats

The accumulation of abnormal polyglutamine-containing protein aggregates within the cytosol and nuclei of affected neurons is a hallmark of the progressive neurodegenerative disorders caused by an elongated (CAG)(n) repeat in the genome. The polyglutamine domains are excellent substrates for the enzyme transglutaminase type 2 (tissue), resulting in the formation of cross-links with polypeptides containing lysyl groups. Enzymatic activity toward the Q(n) domains increases greatly upon lengthening of such Q(n) stretches (n > 40). Among the possible amine donors, the glycolytic enzyme glyceraldehyde-3-phosphate-dehydrogenase was shown to tightly bind several proteins involved in polyglutamine expansion diseases. Recently, the authors have shown that K191, K268, and K331, out of the 26 lysines present in glyceraldehyde-3-phosphate-dehydrogenase, are the reactive amine-donor sites forming cross-links with substance P, which bears the simplest Q(n) domain (n = 2). The present study reports that synthetic peptides of both pathological and nonpathological length (n = 43 and 17, respectively) form cross-links with the same K residues located in the C-terminal region of glyceraldehyde-3-phosphate-dehydrogenase. In addition, it is shown that extra K residues present in the C termini of glyceraldehyde-3-phosphate-dehydrogenase are susceptible to cross-linking in the presence of transglutaminase. The present results indicate a possible modulating effect of Q(n) stretches on tissue transglutaminase substrate specificity and mechanism of recognition.     

The composition of the polyglutamine‐containing proteins influences their co‐aggregation properties

The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptides containing expanded polyglutamine tracts has been proposed to be the key mechanism responsible for the abnormal cell functioning in the so‐called polyglutamine diseases. To evaluate to what extent the ability of polyglutamine sequences to recruit other proteins into the intracellular aggregates depends on the composition of the aggregating peptide, we analysed the co‐aggregation properties of the N‐terminal fragment of huntingtin fused with unrelated non‐aggregating and/or self‐aggregating peptides. We show that the ability of the mutated N‐terminal huntingtin fragment to sequester non‐related proteins can be significantly increased by fusion with the non‐aggregating reporter protein [GFP (green fluorescence protein)]. By contrast, fusion with the self‐aggregating C‐terminal fragment of the CFTR (cystic fibrosis transmembrane conductance regulator) dramatically reduces the sequestration of related non‐fused huntingtin fragments. We also demonstrate that the co‐aggregation of different non‐fused N‐terminal huntingtin fragments depends on their length, with long fragments of the wild‐type huntingtin not only excluded from the nuclear inclusions, but also very inefficiently sequestered into the cytoplasmic aggregates formed by the short fragments of mutant protein. Additionally, our results suggest that atypical intracellular aggregation patterns, which include unusual distribution and/or morphology of protein aggregates, are associated with altered ability of accumulating proteins to co‐aggregate with other peptides.     

Modulating huntingtin half-life alters polyglutamine-dependent aggregate formation and cell toxicity

A common finding among the expanded polyglutamine disorders is intracellular protein aggregates. Although the precise role of these aggregates in the disease process is unclear, they are generally ubiquitinated, implicating the ubiquitin-proteasome pathway in neuronal degeneration. To investigate the mechanism of aggregate formation, we have developed a cell culture model to express huntingtin designed to have an altered degradation rate through the ubiquitin-dependent N-end rule pathway. We fused the first 171 amino acids of huntingtin, containing either a pathogenic or normal polyglutamine tract, to the enhanced green fluorescent protein (EGFP). The half-life of soluble huntingtin-EGFP was dependent on the degradation signal and the polyglutamine tract length. However, once huntingtin-EGFP with a pathogenic tract had aggregated, the protein was extremely stable. Huntingtin-EGFP with a pathogenic glutamine tract and a shorter half-life displayed a delayed onset of aggregate formation and was more toxic to transfected cells. These data suggest that rapid clearance through the ubiquitin-proteasome pathway slows aggregate formation, yet increases cellular toxicity. Polyglutamine-induced neurotoxicity may therefore be triggered by non-aggregated protein, and aggregate formation itself may be a cellular defense mechanism.     

Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington's disease

Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease (HD). Previously, we have shown that mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. In this study we have investigated the co-aggregation of mutant and wild-type htt fragments. We found that mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. Our data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis.     

Efficient induction of nuclear aggresomes by specific single missense mutations in the DNA-binding domain of a viral AP-1 homolog

Nuclear aggresomes induced by proteins containing an expanded polyglutamine (polyQ) tract are pathologic hallmarks of certain neurodegenerative diseases. Some GFP fusion proteins lacking a polyQ tract may also induce nuclear aggresomes in cultured cells. Here we identify single missense mutations within the basic DNA recognition region of Bam HI Z E B virus replication activator (ZEBRA), an Epstein-Barr virus (EBV)-encoded basic zipper protein without a polyQ tract, that efficiently induced the formation of nuclear aggresomes. Wild-type (WT) ZEBRA was diffusely distributed within the nucleus. Four non-DNA-binding mutants, Z(R179E), Z(R183E), Z(R190E), and Z(K178D) localized to the periphery of large intranuclear spheres, to discrete nuclear aggregates, and to the cytoplasm. Other non-DNA-binding mutants, Z(N182K), Z(N182E), and Z(S186E), did not exhibit this phenotype. The interior of the spheres contained promyelocytic leukemia and HSP70 proteins. ZEBRA mutants directly induced the nuclear aggresome pathway in cells with and without EBV. Specific cellular proteins (SC35 and HDAC6) and viral proteins (WT ZEBRA, Rta, and BMLF1) but not other cellular or viral proteins were recruited to nuclear aggresomes. Co-transfection of WT ZEBRA with aggresome-inducing mutants Z(R183E) and Z(R179E) inhibited late lytic viral protein expression and lytic viral DNA amplification. This is the first reported instance in which nuclear aggresomes are induced by single missense mutations in a viral or cellular protein. We discuss conformational changes in the mutant viral AP-1 proteins that may lead to formation of nuclear aggresomes.     

Does tissue transglutaminase play a role in Huntington's disease?

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.     

The two-stage pathway of ataxin-3 fibrillogenesis involves a polyglutamine-independent step

The aggregation of ataxin-3 is associated with spinocerebellar ataxia type 3, which is characterized by the formation of intraneuronal aggregates. However, the mechanism of aggregation is currently not well understood. Ataxin-3 consists of a folded Josephin domain followed by two ubiquitin-interacting motifs and a C-terminal polyglutamine tract, which in the non-pathological form is less than 45 residues in length. We demonstrate that ataxin-3 with 64 glutamines (at(Q64)) undergoes a two-stage aggregation. The first stage involves formation of SDS-soluble aggregates, and the second stage results in formation of SDS-insoluble aggregates via the poly(Q) region. Both these first and second stage aggregates display typical amyloid-like characteristics. Under the same conditions at(Q15) and at(QHQ) undergo a single step aggregation event resulting in SDS-soluble aggregates, which does not involve the polyglutamine tract. These aggregates do not convert to the SDS-insoluble form. These observations demonstrate that ataxin-3 has an inherent capacity to aggregate through its non-polyglutamine domains. However, the presence of a pathological length polyglutamine tract introduces an additional step resulting in formation of a highly stable amyloid-like aggregate.     

Mammalian, yeast, bacterial, and chemical chaperones reduce aggregate formation and death in a cell model of oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is characterized pathologically by intranuclear inclusions in skeletal muscles and is caused by the expansion of a 10-alanine stretch to 12-17 alanines in the intranuclear poly(A)-binding protein 2 (PABP2). Whereas PABP2 is a major component of the inclusions in OPMD, the pathogenic mechanisms causing disease are unknown. Here we show that polyalanine expansions in PABP2 cause increased numbers of inclusions and enhance death in COS-7 cells. We observed similar increases of protein aggregation and cell death with nuclear-targeted green fluorescent protein linked to longer versus shorter polyalanine stretches. Intranuclear aggregates in our OPMD cell model were associated with heat shock protein (HSP) 40 (HDJ-1) and HSP70. Human HDJ-1, yeast hsp104, a bacterially derived GroEL minichaperone, and the chemical chaperone Me(2)SO reduced both aggregation and cell death in our OPMD model without affecting the levels of PABP2, and similar trends were seen with green fluorescent protein with long polyalanine stretches. Thus, polyalanine expansion mutations in different protein contexts cause proteins to misfold/aggregate and kill cells. The situation in OPMD appears to have many parallels with polyglutamine diseases, raising the possibility that misfolded, aggregate-prone proteins may perturb similar pathways, irrespective of the nature of the mutation or protein context.