Modeling Huntington disease in yeast: Perspectives and future directions

Yeast have been extensively used to model aspects of protein folding diseases, yielding novel mechanistic insights and identifying promising candidate therapeutic targets. In particular, the neurodegenerative disorder Huntington disease (HD), which is caused by the abnormal expansion of a polyglutamine tract in the huntingtin (htt) protein, has been widely studied in yeast. This work has led to the identification of several promising therapeutic targets and compounds that have been validated in mammalian cells, Drosophila and rodent models of HD. Here we discuss the development of yeast models of mutant htt toxicity and misfolding, as well as the mechanistic insights gleaned from this simple model. The role of yeast prions in the toxicity/misfolding of mutant htt is also highlighted. Furthermore, we provide an overview of the application of HD yeast models in both genetic and chemical screens, and the fruitful results obtained from these approaches. Finally, we discuss the future of yeast in neurodegenerative research, in the context of HD and other diseases.     

Modeling Huntington disease in yeast: Perspectives and future directions

Yeast have been extensively used to model aspects of protein folding diseases, yielding novel mechanistic insights and identifying promising candidate therapeutic targets. In particular, the neurodegenerative disorder Huntington disease (HD), which is caused by the abnormal expansion of a polyglutamine tract in the huntingtin (htt) protein, has been widely studied in yeast. This work has led to the identification of several promising therapeutic targets and compounds that have been validated in mammalian cells, Drosophila and rodent models of HD. Here we discuss the development of yeast models of mutant htt toxicity and misfolding, as well as the mechanistic insights gleaned from this simple model. The role of yeast prions in the toxicity/misfolding of mutant htt is also highlighted. Furthermore, we provide an overview of the application of HD yeast models in both genetic and chemical screens, and the fruitful results obtained from these approaches. Finally, we discuss the future of yeast in neurodegenerative research, in the context of HD and other diseases.Key words: Huntington disease, yeast, neurodegeneration, genetic modifiers, prionsThe single-celled eukaryote Saccharomyces cerevisiae has long been involved with the technological advancement of mankind. Commonly known as baker''s yeast, for millennia this organism has been employed for the requisite fermentation in the production of bread, wine, beer and other food products.¹ Louis Pasteur first described the critical role of yeast in fermentation in 1860, and conclusively showed that living yeast cells are required for this process.² Since this time, yeast have been used extensively in biological sciences to explore the fundamental properties of the cell, and have become a vital genetic weapon in the arsenal of modern day medical scientists. This review provides an overview of the development, characterization and utilization of yeast models of Huntington disease (HD). These simple models have provided striking insights into the mechanisms underlying cellular toxicity in this disease, and have also uncovered many promising candidate drug targets for HD, several of which have been validated in animal models and hold great therapeutic promise.     

Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice

Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.     

Impact of the core components of the phosphoenolpyruvate-carbohydrate phosphotransferase system, HPr and EI, on differential protein expression in Ralstonia eutropha H16

In Ralstonia eutropha H16, seven genes encoding proteins being involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified. In order to provide more insights into the poly(3-hydroxybutyrate) (PHB)-leaky phenotype of the HPr/EI deletion mutants H16ΔptsH, H16ΔptsI, and H16ΔptsHI when grown on the non-PTS substrate gluconate, parallel fermentations for comparison of their growth behavior were performed. Samples from the exponential, the early stationary, and late stationary growth phases were investigated by microscopy, gas chromatography and (phospho-) proteome analysis. A total of 71 differentially expressed proteins were identified using 2D-PAGE, Pro-Q Diamond and Coomassie staining, and MALDI-TOF analysis. Detected proteins were classified into five major functional groups: carbon metabolism, energy metabolism, amino acid metabolism, translation, and membrane transport/outer membrane proteins. Proteome analyses revealed enhanced expression of proteins involved in the Entner-Doudoroff pathway and in subsequent reactions in cells of strain H16 compared to the mutant H16ΔptsHI. Furthermore, proteins involved in PHB accumulation showed increased abundance in the wild-type. This expression pattern allowed us to identify proteins affecting carbon metabolism/PHB biosynthesis in strain H16 and translation/amino acid metabolism in strain H16ΔptsHI, and to gain insight into the molecular response of R. eutropha to the deletion of HPr/EI.     

Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH(2)-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH(2)-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.     

Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo

We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt. YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease. Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-). YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes. The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels. YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death. Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt. YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene. These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt.     

A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.     

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.     

Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity

Although NH2-terminal mutant huntingtin (htt) fragments cause neurological disorders in Huntington's disease (HD), it is unclear how toxic htt fragments are generated and contribute to the disease process. Here, we report that complex NH2-terminal mutant htt fragments smaller than the first 508 amino acids were generated in htt-transfected cells and HD knockin mouse brains. These fragments constituted neuronal nuclear inclusions and appeared before neurological symptoms. The accumulation and aggregation of these htt fragments were associated with an age-dependent decrease in proteasome activity and were promoted by inhibition of proteasome activity. These results suggest that decreased proteasome activity contributes to late onset htt toxicity and that restoring the ability to remove NH2-terminal fragments will provide a more effective therapy for HD than inhibiting their production.     

Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model

Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins.     

Hsp70 and Hsp40 Functionally Interact with Soluble Mutant Huntingtin Oligomers in a Classic ATP-dependent Reaction Cycle

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.     

Type 2 transglutaminase differentially modulates striatal cell death in the presence of wild type or mutant huntingtin

Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt), is characterized by extensive loss of striatal neurons. The dysregulation of type 2 transglutaminase (TG2) has been proposed to contribute to the pathogenesis in HD as TG2 is up-regulated in HD brain and knocking out TG2 in mouse models of HD ameliorates the disease process. To understand the role of TG2 in the pathogenesis of HD, immortalized striatal cells established from mice in which mutant htt with a polyglutamine stretch of 111 Gln had been knocked-in and wild type (WT) littermates, were stably transfected with human TG2 in a tetracycline inducible vector. Overexpression of TG2 in the WT striatal cells resulted in significantly greater cell death under basal conditions as well as in response to thapsigargin treatment, which causes increased intracellular calcium concentrations. Furthermore, in WT striatal cells TG2 overexpression potentiated mitochondrial membrane depolarization, intracellular reactive oxygen species production, and apoptotic cell death in response to thapsigargin. In contrast, in mutant striatal cells, TG2 overexpression did not increase cell death, nor did it potentiate thapsigargin-induced mitochondrial membrane depolarization or intracellular reactive oxygen species production. Instead, TG2 overexpression in mutant striatal cells attenuated the thapsigargin-activated apoptosis. When in situ transglutaminase activity was quantitatively analyzed in these cell lines, we found that in response to thapsigargin treatment TG2 was activated in WT, but not mutant striatal cells. These data suggest that mutant htt alters the activation of TG2 in response to certain stimuli and therefore differentially modulates how TG2 contributes to cell death processes.     

Dendritic spine loss and neurodegeneration is rescued by Rab11 in models of Huntington's disease

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein (htt) that mediates formation of intracellular protein aggregates. In the brains of HD patients and HD transgenic mice, accumulation of protein aggregates has been causally linked to lesions in axo-dendritic and synaptic compartments. Here we show that dendritic spines - sites of synaptogenesis - are lost in the proximity of htt aggregates because of functional defects in local endosomal recycling mediated by the Rab11 protein. Impaired exit from recycling endosomes (RE) and association of endocytosed protein with intracellular structures containing htt aggregates was demonstrated in cultured hippocampal neurons cells expressing a mutant htt fragment. Dendrites in hippocampal neurons became dystrophic around enlarged amphisome-like structures positive for Rab11, LC3 and mutant htt aggregates. Furthermore, Rab11 overexpression rescues neurodegeneration and dramatically extends lifespan in a Drosophila model of HD. Our findings are consistent with the model that mutant htt aggregation increases local autophagic activity, thereby sequestering Rab11 and diverting spine-forming cargo from RE into enlarged amphisomes. This mechanism may contribute to the toxicity caused by protein misfolding found in a number of neurodegenerative diseases.     

Native mutant huntingtin in human brain: evidence for prevalence of full-length monomer

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.     

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.     

Protective up-regulation of CK2 by mutant huntingtin in cells co-expressing NMDA receptors

Huntington's disease is caused by a polyglutamine expansion in the huntingtin (htt) protein, and previous data indicate that over-activation of NMDA receptors (NMDARs) may be involved in the selective degeneration of cells expressing NR1/NR2B NMDARs. We used Kinetworks™ multi-immunoblotting screens to examine expression of 76 protein kinases, 18 protein phosphatases, 25 heat shock/stress proteins, and 27 apoptosis proteins in human embryonic kidney 293 cells transfected with NR1/NR2B and htt containing 15 (htt-15Q; wild-type) or 138 (htt-138Q; mutant) glutamine repeats. Follow-up experiments revealed several proteins involved in the heat-shock response pathway to be up-regulated in the soluble fraction from cells expressing htt-138Q, including protein phosphatase 5 and cyclin-dependent kinase 5. Increased expression in the soluble fraction of htt-138Q-expressing cells was also noted for the stress- and calcium-activated protein-serine/threonine kinase casein kinase 2, a change which was confirmed in striatal tissue of yeast artificial chromosome transgenic mice expressing full-length mutant htt. Inhibition of casein kinase 2 activity in cultured striatal neurons from these mice significantly exacerbated NMDAR-mediated toxicity, as assessed by labeling of apoptotic nuclei. Our findings are consistent with up-regulation of components of the stress response pathway in the presence of polyglutamine-expanded htt and NR1/NR2B which may reflect an attempt at the cellular level to ameliorate the detrimental effects of mutant htt expression.     

Sex-dependent effect of BAG1 in ameliorating motor deficits of Huntington disease transgenic mice

The pathogenesis of Huntington disease (HD) is attributed to the misfolding of huntingtin (htt) caused by an expanded polyglutamine (polyQ) domain. Considerable effort has been devoted to identifying molecules that can prevent or reduce htt misfolding and the associated neuropathology. Although overexpression of chaperones is known to reduce htt cytotoxicity in cellular models, only modest protection is seen with Hsp70 overexpression in HD mouse models. Because the activity of Hsp70 is modulated by co-chaperones, an interesting issue is whether the in vivo effects of chaperones on polyQ protein toxicity are dependent on other modulators. In the present study, we focused on BAG1, a co-chaperone that interacts with Hsp70 and regulates its activity. Of htt mice expressing the N171-82Q mutant, we found that male N171-82Q mice show a greater deficit in rotarod performance than female N171-82Q mice. This sex-dependent motor deficit was improved by crossing N171-82Q mice with transgenic mice overexpressing BAG1 in neurons. Transgenic BAG1 also reduces the levels of mutant htt in synaptosomal fraction of male HD mice. Overexpression of BAG1 augmented the effects of Hsp70 by reducing aggregation of mutant htt in cultured cells and improving neurite outgrowth in htt-transfected PC12 cells. These findings suggest that the effects of chaperones on HD pathology are influenced by both their modulators and sex-dependent factors.     

Synaptic mutant huntingtin inhibits synapsin-1 phosphorylation and causes neurological symptoms

Many genetic mouse models of Huntington’s disease (HD) have established that mutant huntingtin (htt) accumulates in various subcellular regions to affect a variety of cellular functions, but whether and how synaptic mutant htt directly mediates HD neuropathology remains to be determined. We generated transgenic mice that selectively express mutant htt in the presynaptic terminals. Although it was not overexpressed, synaptic mutant htt caused age-dependent neurological symptoms and early death in mice as well as defects in synaptic neurotransmitter release. Mass spectrometry analysis of synaptic fractions and immunoprecipitation of synapsin-1 from HD CAG150 knockin mouse brains revealed that mutant htt binds to synapsin-1, a protein whose phosphorylation is critical for neurotransmitter release. We found that polyglutamine-expanded exon1 htt binds to the C-terminal region of synapsin-1 to reduce synapsin-1 phosphorylation. Our findings point to a critical role for synaptic htt in the neurological symptoms of HD, providing a new therapeutic target.