Polyglutamine (polyQ) expansion within the ataxin-7 protein, a member of the STAGA [SPT3-TAF(II)31-GCN5L acetylase] and TFTC (GCN5 and TRRAP) chromatin remodeling complexes, causes the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7). Proteolytic processing of ataxin-7 by caspase-7 generates N-terminal toxic polyQ-containing fragments that accumulate with disease progression and play an important role in SCA7 pathogenesis. To elucidate the basis for the toxicity of these fragments, we evaluated which posttranslational modifications of the N-terminal fragment of ataxin-7 modulate turnover and toxicity. Here, we show that mutating lysine 257 (K257), an amino acid adjacent to the caspase-7 cleavage site of ataxin-7 regulates turnover of the truncation product in a repeat-dependent manner. Modification of ataxin-7 K257 by acetylation promotes accumulation of the fragment, while unmodified ataxin-7 is degraded. The degradation of the caspase-7 cleavage product is mediated by macroautophagy in cell culture and primary neuron models of SCA7. Consistent with this, the fragment colocalizes with autophagic vesicle markers, and enhanced fragment accumulation increases in these lysosomal structures. We suggest that the levels of fragment accumulation within the cell is a key event in SCA7 neurodegeneration, and enhancing clearance of polyQ-containing fragments may be an effective target to reduce neurotoxicity in SCA7.
SUMMARY Huntington’s Disease (HD) is characterized by a mutation in the huntingtin gene encoding an expansion of glutamine repeats on the N-terminus of the huntingtin (Htt) protein. Numerous studies have identified Htt proteolysis as a critical pathological event in post mortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD targets. We report the use of the substrate activity screening method against caspases-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models, these irreversible inhibitors suppressed Hdh111Q/111Q-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development.
Huntington's disease (HD) 1 is a hereditary neurodegenerative disorder characterized by unrestrained movements, emotional disturbances, and psychological deterioration (1). HD patients suffer neuronal degeneration in the striatum and frontal and temporal cortex (2). HD is caused by the expansion of a polyglutamine (polyQ) stretch within the huntingtin protein (Htt). Under normal conditions, Htt is a protein with a polyQ stretch containing 2-34 repeats whereas the disease form of Htt has a polyQ repeat length longer than 36 -37. Mechanisms of toxicity for mutant Htt include proteolytic cleavage by proteases such as caspase to produce toxic fragments, impaired vesicular transport, and altered transcription by binding with specific transcription coactivators and transcription factors (3-13).Previous studies show that Htt undergoes post-translational modifications (PTMs), which are associated with alterations in localization or conformation of Htt and regulate mutant Htt toxicity (14 -17). For example, phosphorylation of Htt at Ser-421 by protein kinase Akt1 can protect striatal neurons against mutant Htt-induced toxicity (18). We previously completed an analysis by MS of the phosphorylation of full-length Htt identifying numerous phosphorylation sites throughout the 3144 amino acid sequence of Htt. One of the sites was the phosphorylation at Ser-536, which blocked Htt calpain proteolysis and toxicity (14,19,20). In addition, lack of phosphorylation at serine-1181 Ser-1181 and Ser-1201 and serine-1201 by cyclin-dependent kinase 5 (Cdk5) leads to toxicity and accelerated neuron death (21,22). Other PTMs such as SUMOylation (23) and ubiquitination (24) occur in Htt and alter cellular toxicity and turnover.Acetylation is a covalent reaction in which an acetyl group is introduced to the free amino group of the protein N-terminus or the -amino group of lysine residues. Similar to phosphorylation, acetylation is reversible and highly regulated by histone acetyltransferases and histone deacetylases (HDACs) (25-28). Because lysine acetylation in proteins is involved in many biological functions including transcriptional regulation (29 -31), apoptosis (32-34), energy metabolism (35), and DNA-related activities (36 -44), there is increasing interest in exploring protein acetylation in vitro and in vivo. As compared with phosphorylation, lysine acetylation of substrates in mammalian systems generally have less specificity for the residues surrounding acetyllysine residues making predictions of acetylation sites less reliable (35,45). To date the only estab-
SUMMARY Huntington’s disease (HD) is caused by a mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats at the N-terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knockout mouse (Casp6−/−) Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6−/− mice, regardless of HD genotype. We predicted the Casp6−/− mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6−/− mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6−/− mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 amino acid Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.
A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.
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