ABSTRACT
Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.
Subject(s)
Huntington Disease/pathology , Mutant Proteins/genetics , Mutant Proteins/toxicity , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/toxicity , Nuclear Proteins/genetics , Nuclear Proteins/toxicity , Peptides , Animals , Huntingtin Protein , Mutant Proteins/chemistry , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Protein Conformation , Protein Multimerization , RatsABSTRACT
The use of genetic screens to define cellular pathways that regulate neurodegenerative disease proteins has emerged as a powerful strategy to identify potential therapeutic targets for these disorders. Using cross-species genetic screens, Park et al. recently identified RAS-MAPK-MSK1 as a cellular pathway that modulates levels of the polyglutamine-containing protein ATXN1 and its subsequent toxicity in SCA1.
Subject(s)
Drosophila melanogaster/metabolism , Mitogen-Activated Protein Kinases/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Spinocerebellar Ataxias/metabolism , Spinocerebellar Ataxias/pathology , ras Proteins/metabolism , Animals , Female , Humans , MaleABSTRACT
Many neurodegenerative disorders, such as Alzheimer's, Parkinson's and polyglutamine diseases, share a common pathogenic mechanism: the abnormal accumulation of disease-causing proteins, due to either the mutant protein's resistance to degradation or overexpression of the wild-type protein. We have developed a strategy to identify therapeutic entry points for such neurodegenerative disorders by screening for genetic networks that influence the levels of disease-driving proteins. We applied this approach, which integrates parallel cell-based and Drosophila genetic screens, to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of a polyglutamine tract in ataxin 1 (ATXN1). Our approach revealed that downregulation of several components of the RAS-MAPK-MSK1 pathway decreases ATXN1 levels and suppresses neurodegeneration in Drosophila and mice. Importantly, pharmacological inhibitors of components of this pathway also decrease ATXN1 levels, suggesting that these components represent new therapeutic targets in mitigating SCA1. Collectively, these data reveal new therapeutic entry points for SCA1 and provide a proof-of-principle for tackling other classes of intractable neurodegenerative diseases.
Subject(s)
Drosophila melanogaster/metabolism , Mitogen-Activated Protein Kinases/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Spinocerebellar Ataxias/metabolism , Spinocerebellar Ataxias/pathology , ras Proteins/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Ataxin-1 , Ataxins , Cell Line, Tumor , Disease Models, Animal , Down-Regulation/drug effects , Drosophila melanogaster/genetics , Female , Humans , MAP Kinase Signaling System/drug effects , Male , Mice , Molecular Sequence Data , Molecular Targeted Therapy , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphorylation , Protein Stability/drug effects , Ribosomal Protein S6 Kinases, 90-kDa/deficiency , Ribosomal Protein S6 Kinases, 90-kDa/genetics , TransgenesABSTRACT
Mitochondrial dysfunction and elevated reactive oxygen species are strongly implicated in both aging and various neurodegenerative disorders, including Huntington disease (HD). Because reactive oxygen species can promote the selective oxidation of protein cysteine sulfhydryl groups to disulfide bonds we examined the spectrum of disulfide-bonded proteins that were specifically altered in a HD context. Protein extracts from PC12 cells overexpressing the amino-terminal fragment of the Huntingtin (Htt) protein with either a nonpathogenic or pathogenic polyglutamine repeat (Htt-103Q) were resolved by redox two-dimensional PAGE followed by mass spectrometry analysis. Several antioxidant proteins were identified that exhibited changes in disulfide bonding unique to Htt-103Q expressing cells. In particular, the antioxidant protein peroxiredoxin 1 (Prx1) exhibited both decreased expression and hyperoxidation in response to mutant Htt expressed in either PC12 cells or immortalized striatal cells exposed to 3-nitropropionic acid. Ectopic expression of Prx1 in PC12 cells attenuated mutant Htt-induced toxicity. In contrast, short hairpin RNA-mediated knockdown of Prx1 potentiated mHtt toxicity. Furthermore, treatment with the dithiol-based compounds dimercaptopropanol and dimercaptosuccinic acid suppressed toxicity in both HD cell models, whereas monothiol compounds were relatively ineffective. Dimercaptopropanol treatment also prevented mutant Htt-induced loss of Prx1 expression in both cell models. Our studies reveal for the first time that pathogenic Htt can affect the expression and redox state of antioxidant proteins; an event countered by specific dithiol-based compounds. These findings should provide a catalyst to explore the use of dithiol-based drugs for the treatment of neurodegenerative diseases.
Subject(s)
Huntington Disease/drug therapy , Nerve Degeneration/drug therapy , Nerve Tissue Proteins/genetics , Neuroprotective Agents/pharmacology , Nuclear Proteins/genetics , Peroxiredoxins/metabolism , Toluene/analogs & derivatives , Animals , Cell Death/drug effects , Cell Line, Transformed , Corpus Striatum/cytology , Disulfides/metabolism , Huntingtin Protein , Huntington Disease/genetics , Huntington Disease/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Oxidative Stress/drug effects , PC12 Cells , Peptides/metabolism , Peroxiredoxins/genetics , RNA, Small Interfering/genetics , Rats , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Toluene/pharmacologyABSTRACT
Huntington's disease (HD) is caused by a polyglutamine expansion in the Huntingtin (Htt) protein. Proteolytic cleavage of Htt into toxic N-terminal fragments is believed to be a key aspect of pathogenesis. The best characterized putative cleavage event is at amino acid 586, hypothesized to be mediated by caspase 6. A corollary of the caspase 6 cleavage hypothesis is that the caspase 6 fragment should be a toxic fragment. To test this hypothesis, and further characterize the role of this fragment, we have generated transgenic mice expressing the N-terminal 586 aa of Htt with a polyglutamine repeat length of 82 (N586-82Q), under the control of the prion promoter. N586-82Q mice show a clear progressive rotarod deficit by 4 months of age, and are hyperactive starting at 5 months, later changing to hypoactivity before early mortality. MRI studies reveal widespread brain atrophy, and histologic studies demonstrate an abundance of Htt aggregates, mostly cytoplasmic, which are predominantly composed of the N586-82Q polypeptide. Smaller soluble N-terminal fragments appear to accumulate over time, peaking at 4 months, and are predominantly found in the nuclear fraction. This model appears to have a phenotype more severe than current full-length Htt models, but less severe than HD mouse models expressing shorter Htt fragments. These studies suggest that the caspase 6 fragment may be a transient intermediate, that fragment size is a factor contributing to the rate of disease progression, and that short soluble nuclear fragments may be most relevant to pathogenesis.
Subject(s)
Caspase 6/physiology , Huntington Disease/metabolism , Nerve Degeneration/metabolism , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Peptide Fragments/genetics , Animals , Atrophy , Disease Models, Animal , Humans , Huntingtin Protein , Huntington Disease/pathology , Huntington Disease/physiopathology , Mice , Mice, Inbred Strains , Mice, Transgenic , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Peptide Fragments/biosynthesis , Peptide Fragments/toxicity , Trinucleotide Repeat Expansion/physiologyABSTRACT
BACKGROUND: The 90-kDa ribosomal S6 kinase (Rsk) family is involved in cell survival. Rsk activation is regulated by sequential phosphorylations controlled by extracellular signal-regulated kinase (ERK) 1/2 and 3-phosphoinositide-dependent protein kinase 1 (PDK1). Altered ERK1/2 and PDK1 phosphorylation have been described in Huntington's disease (HD), characterized by the expression of mutant huntingtin (mhtt) and striatal degeneration. However, the role of Rsk in this neurodegenerative disease remains unknown. Here, we analyzed the protein levels, activity and role of Rsk in in vivo and in vitro HD models. RESULTS: We observed increased protein levels of Rsk1 and Rsk2 in the striatum of Hdh(Q111/Q111) and R6/1 mice, STHdh(Q111/Q111) cells and striatal cells transfected with full-length mhtt. Analysis of the phosphorylation of Rsk in Hdh mice and STHdh cells showed reduced levels of phospho Ser-380 (dependent on ERK1/2), whereas phosphorylation at Ser-221 (dependent on PDK1) was increased. Moreover, we found that elevated Rsk activity in STHdh(Q111/Q111) cells was mainly due to PDK1 activity, as assessed by transfection with Rsk mutant constructs. The increase of Rsk in STHdh(Q111/Q111) cells occurred in the cytosol and in the nucleus, which results in enhanced phosphorylation of both cytosolic and nuclear Rsk targets. Finally, pharmacological inhibition of Rsk, knock-down and overexpression experiments indicated that Rsk activity exerts a protective effect against mhtt-induced cell death in STHdh(Q7/Q7) cells transfected with mhtt. CONCLUSION: The increase of Rsk levels and activity would act as a compensatory mechanism with capacity to prevent mhtt-mediated cell death. We propose Rsk as a good target for neuroprotective therapies in HD.
Subject(s)
Huntington Disease/physiopathology , Nerve Tissue Proteins/toxicity , Nuclear Proteins/toxicity , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Animals , Cells, Cultured , Corpus Striatum/cytology , Corpus Striatum/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Huntingtin Protein , Huntington Disease/pathology , Male , Mice , Mice, Transgenic , Mutation , Phosphorylation , Ribosomal Protein S6 Kinases, 90-kDa/geneticsABSTRACT
Huntington disease (HD) is caused by the expansion of polyglutamine (polyQ) repeats in the amino-terminal of hungtintin (htt). PolyQ-expanded htt forms intracellular ubiquitinated aggregates in neurons and causes neuronal cell death. Here, utilizing a HD cellular model, we report that Tollip, an ubiquitin binding protein that participates in intracellular transport via endosomes, co-localizes with and stimulates aggregation of polyQ-expanded amino-terminal htt. Furthermore, we demonstrate that Tollip protects cells against the toxicity of polyQ-expanded htt. We propose that association of Tollip with polyubiquitin accelerates aggregation of toxic htt species into inclusions and thus provides a cell protective role by sequestration.
Subject(s)
Intracellular Signaling Peptides and Proteins/pharmacology , Nerve Tissue Proteins/toxicity , Neurons/drug effects , Neuroprotective Agents , Nuclear Proteins/toxicity , Peptides/antagonists & inhibitors , Peptides/toxicity , Ubiquitin/metabolism , Cell Death/drug effects , Cell Line , Endosomes/metabolism , Fluorescent Antibody Technique , Green Fluorescent Proteins , HEK293 Cells , Humans , Huntingtin Protein , Immunohistochemistry , Indoles , Nerve Tissue Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Qa-SNARE Proteins/metabolism , RNA Interference , Serine Proteinase Inhibitors , Vesicular Transport Proteins/metabolismABSTRACT
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: bna4Δ, mbf1Δ, and ume1Δ. 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.
Subject(s)
Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Gene Expression Profiling , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Protein Biosynthesis , Saccharomyces cerevisiae/genetics , Base Sequence , Gene Deletion , Genomics , Humans , Huntingtin Protein , Huntington Disease/genetics , Mutant Proteins/biosynthesis , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutant Proteins/toxicity , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Open Reading Frames/genetics , RNA, Ribosomal/genetics , Regulatory Sequences, Nucleic Acid/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/cytology , Transgenes/geneticsABSTRACT
Proteolytic cleavage of huntingtin (Htt) is known to be a key event in the pathogenesis of Huntington's disease (HD). Our understanding of proteolytic processing of Htt has thus far focused on the protease families-caspases and calpains. Identifying critical proteases involved in Htt proteolysis and toxicity using an unbiased approach has not been reported. To accomplish this, we designed a high-throughput western blot-based screen to examine the generation of the smallest N-terminal polyglutamine-containing Htt fragment. We screened 514 siRNAs targeting the repertoire of human protease genes. This screen identified 11 proteases that, when inhibited, reduced Htt fragment accumulation. Three of these belonged to the matrix metalloproteinase (MMP) family. One family member, MMP-10, directly cleaves Htt and prevents cell death when knocked down in striatal Hdh(111Q/111Q) cells. Correspondingly, MMPs are activated in HD mouse models, and loss of function of Drosophila homologs of MMPs suppresses Htt-induced neuronal dysfunction in vivo.
Subject(s)
Huntington Disease/genetics , Matrix Metalloproteinases/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Animals , Caspases/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Line, Transformed , Corpus Striatum/pathology , Disease Models, Animal , Drosophila , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/genetics , Humans , Huntingtin Protein , Matrix Metalloproteinases/classification , Matrix Metalloproteinases/genetics , Mice , Mice, Neurologic Mutants , Mutation/genetics , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , Neurons/drug effects , Neurons/metabolism , Nuclear Proteins/drug effects , Nuclear Proteins/genetics , Peptides/genetics , Peptides/metabolism , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , Transfection/methodsABSTRACT
Spinocerebellar ataxia 1 (SCA1) is a dominantly inherited neurodegenerative disease associated with progressive ataxia resulting from the loss of cerebellar Purkinje cells (PCs) and neurons in the brainstem. In PCs of SCA1 transgenic mice, the disease causing ataxin-1 protein mediates the formation of S100B containing cytoplasmic vacuoles and further self-aggregates to form intranuclear inclusions. The exact function of the ataxin-1 protein is not fully understood. However, the aggregation and neurotoxicity of the mutant ataxin-1 protein is dependent on the phosphorylation at serine 776 (S776). Although protein kinase A (PKA) has been implicated as the S776 kinase, the mechanism of PKA/ataxin-1 regulation in SCA1 is still not clear. We propose that a dopamine D(2) receptor (D2R)/S100B pathway may be involved in modulating PKA activity in PCs. Using a D2R/S100B HEK stable cell line transiently transfected with GFP-ataxin-1[82Q], we demonstrate that stimulation of the D2R/S100B pathway caused a reduction in mutant ataxin-1 S776 phosphorylation and ataxin-1 aggregation. Activation of PKA by forskolin resulted in an enhanced S776 phosphorylation and increased ataxin-1 nuclear aggregation, which was suppressed by treatment with D2R agonist bromocriptine and PKA inhibitor H89. Furthermore, treating SCA1 transgenic PC slice cultures with forskolin induced neurodegenerative morphological abnormalities in PC dendrites consistent with those observed in vivo. Taken together our data support a mechanism where PKA dependent mutant ataxin-1 phosphorylation and aggregation can be regulated by D2R/S100B signaling.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Receptors, Dopamine D2/physiology , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/metabolism , Animals , Ataxin-1 , Ataxins , Cell Line , Cells, Cultured , Dopamine/metabolism , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Mice , Mice, Transgenic , Mutation/genetics , Nerve Growth Factors/physiology , Nerve Tissue Proteins/physiology , Nerve Tissue Proteins/toxicity , Nuclear Proteins/physiology , Nuclear Proteins/toxicity , Organ Culture Techniques , Phosphorylation/genetics , S100 Calcium Binding Protein beta Subunit , S100 Proteins/physiology , Spinocerebellar Ataxias/physiopathologyABSTRACT
Abnormal expansion of a polyglutamine tract in huntingtin (Htt) protein results in Huntington's disease (HD), an autosomal dominant neurodegenerative disorder involving progressive loss of motor and cognitive function. Contrasting with the ubiquitous tissue expression of polyglutamine-expanded Htt, HD pathology is characterized by the increased vulnerability of specific neuronal populations within the striatum and the cerebral cortex. Morphological, biochemical, and functional characteristics of neurons affected in HD that might render these cells more vulnerable to the toxic effects of polyglutamine-Htt are covered in this review. The differential vulnerability of neurons observed in HD is discussed in the context of various major pathogenic mechanisms proposed to date, and in line with evidence showing a 'dying-back' pattern of degeneration in affected neuronal populations.
Subject(s)
Huntington Disease/pathology , Neurons/pathology , Axonal Transport/physiology , Brain/pathology , Brain Chemistry/physiology , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/physiology , Gene Expression/genetics , Gene Expression/physiology , Humans , Huntingtin Protein , Huntington Disease/etiology , Huntington Disease/genetics , Mitochondria/pathology , Mutation/physiology , Nerve Degeneration/pathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/toxicity , Neurons/classification , Nuclear Proteins/genetics , Nuclear Proteins/toxicity , Signal TransductionABSTRACT
Spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by the expansion of the polyglutamine repeat region within the ataxin-3 protein. The mutant protein forms intracellular aggregates in the brain. However, the cellular mechanisms causing toxicity are still poorly understood and there are currently no effective treatments. In this study we show that administration of a rapamycin ester (cell cycle inhibitor-779, temsirolimus) improves motor performance in a transgenic mouse model of spinocerebellar ataxia type 3. Temsirolimus inhibits mammalian target of rapamycin and hence upregulates protein degradation by autophagy. Temsirolimus reduces the number of aggregates seen in the brains of transgenic mice and decreases levels of cytosolic soluble mutant ataxin-3, while endogenous wild-type protein levels remain unaffected. Temsirolimus is designed for long-term use in patients and therefore represents a possible therapeutic strategy for the treatment of spinocerebellar ataxia type 3. Using this disease model and treatment paradigm, we employed a microarray approach to investigate transcriptional changes that might be important in the pathogenesis of spinocerebellar ataxia type 3. This identified ubiquitin specific peptidase-15, which showed expression changes at both the messenger ribonucleic acid and protein level. Ubiquitin specific peptidase-15 levels were also changed in mice expressing another mutant polyglutamine protein, huntingtin. In total we identified 16 transcripts that were decreased in transgenic ataxin-3 mice that were normalized following temsirolimus treatment. In this mouse model with relatively mild disease progression, the number of transcripts changed was low and the magnitude of these changes was small. However, the importance of these transcriptional alterations in the pathogenesis of spinocerebellar ataxia type 3 remains unclear.
Subject(s)
Autophagy/genetics , Disease Models, Animal , Machado-Joseph Disease/genetics , Mutation/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , Ataxin-3 , Autophagy/drug effects , Cells, Cultured , Humans , Machado-Joseph Disease/drug therapy , Machado-Joseph Disease/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nuclear Proteins/biosynthesis , Nuclear Proteins/toxicity , Rats , Rats, Sprague-Dawley , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Sirolimus/therapeutic use , Transcription Factors/biosynthesis , Transcription Factors/toxicityABSTRACT
Huntington's disease (HD) is a devastating neurodegenerative disorder that is inherited in an autosomal dominant fashion and is caused by a polyglutamine expansion in the protein huntingtin (htt). In recent years, modeling of various aspects of HD in the yeast Saccharomyces cerevisiae has provided insight into the conserved mechanisms of mutant htt toxicity in eukaryotic cells. The high degree of conservation of cellular and molecular processes between yeast and mammalian cells have made it a valuable system for studying basic mechanisms underlying human disease. Yeast models of HD recapitulate conserved disease-relevant phenotypes and can be used for drug discovery efforts as well as to gain mechanistic and genetic insights into candidate drugs. Here we provide a detailed overview of yeast models of mutant htt misfolding and toxicity and the molecular and phenotypic characterization of these models. We also review how these models identified novel therapeutic targets and compounds for HD and discuss the benefits and limitations of this model genetic system. Finally, we discuss how yeast may be used to provide further insight into the molecular and cellular mechanisms underlying HD and treatment strategies for this devastating disorder.
Subject(s)
Huntington Disease/genetics , Models, Genetic , Saccharomyces cerevisiae/genetics , Drug Evaluation, Preclinical/methods , Genomics/methods , Humans , Huntingtin Protein , Huntington Disease/etiology , Huntington Disease/therapy , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/toxicity , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/toxicity , Phenotype , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/toxicity , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Trinucleotide Repeat ExpansionABSTRACT
Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg(167). This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg(167) may mediate mutant huntingtin toxicity in Huntington disease.
Subject(s)
Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Neurons/metabolism , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Amino Acid Sequence , Animals , Cell Line , Humans , Huntingtin Protein , Huntington Disease/metabolism , Huntington Disease/pathology , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Neurons/cytology , Nuclear Proteins/genetics , Peptide Fragments/geneticsABSTRACT
Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.
Subject(s)
Animals, Genetically Modified , Brain-Derived Neurotrophic Factor/metabolism , Carbazoles/metabolism , Enzyme Inhibitors/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/toxicity , Neuroprotective Agents/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/toxicity , Animals , Brain-Derived Neurotrophic Factor/genetics , Carbazoles/chemistry , Carbazoles/therapeutic use , Cell Line , Disease Models, Animal , Drosophila melanogaster , Enzyme Activation , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Huntingtin Protein , Huntington Disease/genetics , Huntington Disease/metabolism , Indole Alkaloids/chemistry , Indole Alkaloids/metabolism , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Molecular Structure , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/therapeutic use , Neuroprotective Agents/chemistry , Neuroprotective Agents/therapeutic use , Nuclear Proteins/metabolism , Nuclear Proteins/therapeutic use , Phenotype , RatsABSTRACT
Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.
Subject(s)
Amide Synthases/metabolism , Drosophila Proteins/metabolism , Drosophila/enzymology , Molecular Chaperones/metabolism , Nerve Degeneration , Neurodegenerative Diseases/prevention & control , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Amide Synthases/genetics , Animals , Ataxin-1 , Ataxins , Brain/metabolism , COS Cells , Chlorocebus aethiops , Disease Models, Animal , Drosophila Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , Molecular Chaperones/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/pathology , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Spinocerebellar Ataxias/enzymology , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/pathology , Spinocerebellar Ataxias/prevention & controlABSTRACT
Huntingtin is an antiapoptotic protein that becomes toxic when its polyglutamine stretch is expanded, resulting in Huntington's disease (HD). Protein context and posttranslational modifications regulate huntingtin toxicity. Identifying signaling pathways that act on huntingtin is, therefore, key to understanding huntingtin function in normal and pathological conditions. We show here that huntingtin is phosphorylated by the cyclin-dependent kinase 5 (Cdk5) at serines 1181 and 1201. Phosphorylation can be induced by DNA damage in vitro and in vivo. The state of huntingtin phosphorylation is a crucial regulator of neuronal cell death. Absence of phosphorylation of huntingtin at serines 1181 and 1201 confers toxic properties to wild-type huntingtin in a p53-dependent manner in striatal neurons and accelerates neuronal death induced by DNA damage. In contrast, phosphorylation at serines 1181 and 1201 protects against polyQ-induced toxicity. Finally, we show in late stages of HD a sustained DNA damage that is associated with a decrease in Cdk5/p35 levels. We propose that wild-type huntingtin is a component of the DNA damage response signal in neurons and that the Cdk5/DNA damage pathway is dysregulated in HD.
Subject(s)
Cyclin-Dependent Kinase 5/physiology , DNA Damage/physiology , Mutation , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Neurons/metabolism , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Aged , Aged, 80 and over , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Cells, Cultured , Cyclin-Dependent Kinase 5/genetics , DNA Damage/drug effects , Gene Expression Regulation, Enzymologic/physiology , Humans , Huntingtin Protein , Huntington Disease/enzymology , Huntington Disease/genetics , Huntington Disease/metabolism , Mice , Middle Aged , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Neurons/drug effects , Neurons/enzymology , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Phosphorylation/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.
Subject(s)
Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Animals , Ataxin-1 , Ataxins , Cells, Cultured , Drosophila melanogaster/anatomy & histology , Humans , Intranuclear Inclusion Bodies/metabolism , Molecular Chaperones/metabolism , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/metabolism , Neurons/pathology , Nuclear Proteins/genetics , Peptides/toxicity , Photoreceptor Cells, Invertebrate/cytology , Photoreceptor Cells, Invertebrate/metabolism , Photoreceptor Cells, Invertebrate/pathology , Protein Conformation , Protein Folding , Spinocerebellar Ataxias/metabolism , Spinocerebellar Ataxias/pathology , Transgenes , Ubiquitin-Protein Ligases/geneticsABSTRACT
Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.
Subject(s)
Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/toxicity , Nuclear Proteins/metabolism , Nuclear Proteins/toxicity , Phosphopeptides/metabolism , Phosphopeptides/toxicity , Protein Interaction Mapping , Amino Acid Sequence , Animals , Cell Line , Chromatography, High Pressure Liquid , Humans , Huntingtin Protein , Huntington Disease/metabolism , Hydrolysis , Mitogen-Activated Protein Kinase 1/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/isolation & purification , Nuclear Proteins/isolation & purification , PC12 Cells , Peptide Hydrolases/metabolism , Phosphopeptides/isolation & purification , Phosphorylation , Protein Interaction Mapping/methods , Rats , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Huntington's disease is a neurodegenerative disease caused by an expanded polyglutamine stretch within the huntingtin protein. Transfection of mutant huntingtin causes cell toxicity and depletion of CREB binding protein (CBP) or its recruitment into huntingtin aggregates. However, the role of CBP has been controversial and the relationship between polyglutamine-induced toxicity and CBP depletion has not been examined on an individual cell basis. Using a single-cell based assay, we found that, in HT22 cells or primary neurons transfected with mutant huntingtin, cell toxicity was accompanied by CBP depletion, rather than merely recruitment. Transfection with a htt exon1 construct containing uninterrupted polyglutamine or a polyglutamine region engineered to form a compact beta structure resulted in cell toxicity. CBP depletion was accompanied by histone hypo-acetylation. CBP overexpression rescued both acetylated histone levels and cell toxicity. These data suggest that CBP dysfunction and altered gene transcription contribute to mutant htt-induced neurotoxicity.