Functional gene expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity.
ABSTRACT: Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4?, mbf1?, and ume1?. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.
Project description: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.
Project description:Huntington's disease (HD) is a genetic disease caused by a CAG trinucleotide repeat expansion encoding a polyglutamine tract in the huntingtin (HTT) protein, ultimately leading to neuronal loss and consequent cognitive decline and death. As no treatments for HD currently exist, several chemical screens have been performed using cell-based models of mutant HTT toxicity. These screens measured single disease-related endpoints, such as cell death, but had low 'hit rates' and limited dimensionality for therapeutic detection. Here, we have employed gene expression microarray analysis of HD samples--a snapshot of the expression of 25,000 genes--to define a gene expression signature for HD from publically available data. We used this information to mine a database for chemicals positively and negatively correlated to the HD gene expression signature using the Connectivity Map, a tool for comparing large sets of gene expression patterns. Chemicals with negatively correlated expression profiles were highly enriched for protective characteristics against mutant HTT fragment toxicity in in vitro and in vivo models. This study demonstrates the potential of using gene expression to mine chemical activity, guide chemical screening, and detect potential novel therapeutic compounds.Single-endpoint chemical screens have low therapeutic discovery hit-rates. In the context of HD, we guided a chemical screen using gene expression data. The resulting chemicals were highly enriched for suppressors of mutant HTT fragment toxicity. This study provides a proof of concept for wider usage in all chemical screening.
Project description:Huntington disease is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the protein huntingtin (Htt), which leads to its aggregation in nuclear and cytoplasmic inclusion bodies. We recently identified 52 loss-of-function mutations in yeast genes that enhance the toxicity of a mutant Htt fragment. Here we report the results from a genome-wide loss-of-function suppressor screen in which we identified 28 gene deletions that suppress toxicity of a mutant Htt fragment. The suppressors are known or predicted to have roles in vesicle transport, vacuolar degradation, transcription and prion-like aggregation. Among the most potent suppressors was Bna4 (kynurenine 3-monooxygenase), an enzyme in the kynurenine pathway of tryptophan degradation that has been linked directly to the pathophysiology of Huntington disease in humans by a mechanism that may involve reactive oxygen species. This finding is suggestive of a conserved mechanism of polyglutamine toxicity from yeast to humans and identifies new candidate therapeutic targets for the treatment of Huntington disease.
Project description:Huntington disease (HD) is an inherited neurodegenerative disease caused by a CAG expansion in the HTT gene. Using yeast two-hybrid methods, we identified a large set of proteins that interact with huntingtin (HTT)-interacting proteins. This network, composed of HTT-interacting proteins (HIPs) and proteins interacting with these primary nodes, contains 3235 interactions among 2141 highly interconnected proteins. Analysis of functional annotations of these proteins indicates that primary and secondary HIPs are enriched in pathways implicated in HD, including mammalian target of rapamycin, Rho GTPase signaling, and oxidative stress response. To validate roles for HIPs in mutant HTT toxicity, we show that the Rho GTPase signaling components, BAIAP2, EZR, PIK3R1, PAK2, and RAC1, are modifiers of mutant HTT toxicity. We also demonstrate that Htt co-localizes with BAIAP2 in filopodia and that mutant HTT interferes with filopodial dynamics. These data indicate that HTT is involved directly in membrane dynamics, cell attachment, and motility. Furthermore, they implicate dysregulation in these pathways as pathological mechanisms in HD.
Project description:Huntington's disease (HD) is a fatal neurodegenerative disease caused by mutant huntingtin (htt) protein, and there are currently no effective treatments. Recently, we and others demonstrated that silencing mutant htt via RNA interference (RNAi) provides therapeutic benefit in HD mice. We have since found that silencing wild-type htt in adult mouse striatum is tolerated for at least 4 months. However, given the role of htt in various cellular processes, it remains unknown whether nonallele-specific silencing of both wild-type and mutant htt is a viable therapeutic strategy for HD. Here, we tested whether cosilencing wild-type and mutant htt provides therapeutic benefit and is tolerable in HD mice. After treatment, HD mice showed significant reductions in wild-type and mutant htt, and demonstrated improved motor coordination and survival. We performed transcriptional profiling to evaluate the effects of reducing wild-type htt in adult mouse striatum. We identified gene expression changes that are concordant with previously described roles for htt in various cellular processes. Also, several abnormally expressed transcripts associated with early-stage HD were differentially expressed in our studies, but intriguingly, those involved in neuronal function changed in opposing directions. Together, these encouraging and surprising findings support further testing of nonallele-specific RNAi therapeutics for HD.
Project description:Protein glycation is an age-dependent posttranslational modification associated with several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. By modifying amino-groups, glycation interferes with folding of proteins, increasing their aggregation potential. Here, we studied the effect of pharmacological and genetic manipulation of glycation on huntingtin (HTT), the causative protein in Huntington's disease (HD). We observed that glycation increased the aggregation of mutant HTT exon 1 fragments associated with HD (HTT72Q and HTT103Q) in yeast and mammalian cell models. We found that glycation impairs HTT clearance thereby promoting its intracellular accumulation and aggregation. Interestingly, under these conditions autophagy increased and the levels of mutant HTT released to the culture medium decreased. Furthermore, increased glycation enhanced HTT toxicity in human cells and neurodegeneration in fruit flies, impairing eclosion and decreasing life span. Overall, our study provides evidence that glycation modulates HTT exon-1 aggregation and toxicity, and suggests it may constitute a novel target for therapeutic intervention in HD.
Project description:Although epigenetic abnormalities have been described in Huntington's disease (HD), the causal epigenetic mechanisms driving neurodegeneration in HD cortex and striatum remain undefined. Using an epigenetic pathway-targeted drug screen, we report that inhibitors of DNA methyltransferases (DNMTs), decitabine and FdCyd, block mutant huntingtin (Htt)-induced toxicity in primary cortical and striatal neurons. In addition, knockdown of DNMT3A or DNMT1 protected neurons against mutant Htt-induced toxicity, together demonstrating a requirement for DNMTs in mutant Htt-triggered neuronal death and suggesting a neurodegenerative mechanism based on DNA methylation-mediated transcriptional repression. Inhibition of DNMTs in HD model primary cortical or striatal neurons restored the expression of several key genes, including Bdnf, an important neurotrophic factor implicated in HD. Accordingly, the Bdnf promoter exhibited aberrant cytosine methylation in mutant Htt-expressing cortical neurons. In vivo, pharmacological inhibition of DNMTs in HD mouse brains restored the mRNA levels of key striatal genes known to be downregulated in HD. Thus, disturbances in DNA methylation play a critical role in mutant Htt-induced neuronal dysfunction and death, raising the possibility that epigenetic strategies targeting abnormal DNA methylation may have therapeutic utility in HD.
Project description:Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the Huntingtin (HTT) gene. Proteolytic cleavage of mutant huntingtin (Htt) protein with an expanded polyglutamine (polyQ) stretch results in production of Htt fragments that aggregate and induce impaired ubiquitin proteasome, mitochondrial functioning and transcriptional dysregulation. To understand the time-resolved relationship between aggregate formation and transcriptional changes at early disease stages, we performed temporal transcriptome profiling and quantification of aggregate formation in living cells in an inducible HD cell model.Rat pheochromocytoma (PC12) cells containing a stably integrated, doxycycline-inducible, eGFP-tagged N-terminal human Htt fragment with an expanded polyQ domain were used to analyse gene expression changes at different stages of mutant Htt aggregation. At earliest time points after doxycycline induction no detectable aggregates and few changes in gene expression were observed. Aggregates started to appear at intermediate time points. Aggregate formation and subsequent enlargement of aggregates coincided with a rapid increase in the number of differentially expressed (DE) genes. The increase in number of large aggregates coincided with a decrease in the number of smaller aggregates whereas the transcription profile reverted towards the profile observed before mutant Htt induction. Cluster-based analysis of the 2,176 differentially expressed genes revealed fourteen distinct clusters responding differently over time. Functional enrichment analysis of the two major gene clusters revealed that genes in the up-regulated cluster were mainly involved in metabolic (antioxidant activity and cellular ketone metabolic processes) and genes in the down-regulated cluster in developmental processes, respectively. Promoter-based analysis of the identified gene clusters resulted in identification of a transcription factor network of which several previously have been linked to HD.We demonstrate a time-resolved relationship between Htt aggregation and changes in the transcriptional profile. We identified two major gene clusters showing involvement of (i) mitochondrial dysfunction and (ii) developmental processes implying cellular homeostasis defects. We identified novel and known HD-linked transcription factors and show their interaction with known and predicted regulatory proteins. Our data provide a novel resource for hypothesis building on the role of transcriptional key regulators in early stages of HD and possibly other polyQ-dependent diseases.
Project description:Huntington's disease (HD) is caused in large part by a polyglutamine expansion within the huntingtin (Htt) protein. Post-translational modifications (PTMs) control and regulate many protein functions and cellular pathways, and PTMs of mutant Htt are likely important modulators of HD pathogenesis. Alterations of selected numbers of PTMs of Htt fragments have been shown to modulate Htt cellular localization and toxicity. In this study, we systematically introduced site-directed alterations in individual phosphorylation and acetylation sites in full-length Htt constructs. The effects of each of these PTM alteration constructs were tested on cell toxicity using our nuclear condensation assay and on mitochondrial viability by measuring mitochondrial potential and size. Using these functional assays in primary neurons, we identified several PTMs whose alteration can block neuronal toxicity and prevent potential loss and swelling of the mitochondria caused by mutant Htt. These PTMs included previously described sites such as serine 116 and newly found sites such as serine 2652 throughout the protein. We found that these functionally relevant sites are clustered in protease-sensitive domains throughout full-length Htt. These findings advance our understanding of the Htt PTM code and its role in HD pathogenesis. Because PTMs are catalyzed by enzymes, the toxicity-modulating Htt PTMs identified here may be promising therapeutic targets for managing HD.
Project description:Huntington's disease (HD) is an incurable neurological disorder caused by an abnormal glutamine repeat expansion in the huntingtin (Htt) protein. In the present studies, we investigated the role of Transducers of Regulated cAMP response element-binding (CREB) protein activity (TORCs) in HD, since TORCs play an important role in the expression of the transcriptional co-regulator peroxisome proliferator-activated receptor gamma coactivator 1? (PGC-1?), whose expression is impaired in HD. We found significantly decreased TORC1 expression levels in STHdhQ111 cells expressing mutant Htt, in the striatum of NLS-N171-82Q, R6/2 and HdhQ111 HD transgenic mice and in postmortem striatal tissue from HD patients. TORC1 overexpression in wild-type (WT) and Htt striatal cells increased CREB mRNA and protein levels, PGC-1? promoter activity, mRNA expression of the PGC-1?, NRF-1, Tfam and CytC genes, mitochondrial DNA content, mitochondrial activity and mitochondrial membrane potential. TORC1 overexpression also increased the resistance of striatal cells to 3-nitropropionic (3-NP) acid-mediated toxicity. In cultured WT and mutant Htt striatal cells, small hairpin RNA-mediated TORC1 knockdown resulted in decreased PGC-1? expression and increased susceptibility to 3-NP-induced toxicity. Overexpression of PGC-1? partially prevented TORC1 knockdown-mediated increased susceptibility of Htt striatal cells to 3-NP. Specific knockdown of TORC1 in the striatum of NLS-N171-82Q HD transgenic mice induced neurodegeneration. Lastly, knockdown of Htt prevents transcriptional repression of TORC1 and CREB in Htt striatal cells. These findings show that impaired expression and function of TORC1, which results in a reduction in PGC-1?, plays an important role in mitochondrial dysfunction in HD.