Inhibiting Caspase Cleavage of Huntingtin Reduces Toxicity and Aggregate Formation in Neuronal and Nonneuronal Cells

Abstract

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.Huntington's disease (HD) 1 is a progressive neurodegenerative disorder caused by polyglutamine expansion in the N terminus of htt (1). The cardinal neuropathological feature of HD is the selective neuronal loss of ␥-aminobutyric acid-ergic medium spiny neostriatal neurons and large projection neurons in cortical layers V and VI (2-4). The detection of DNA strand breaks in affected regions of HD patient brains (5-7) suggests that neurodegeneration occurs by an apoptotic mechanism and suggests that caspases could play an important role in HD.Caspases are cysteine aspartic acid proteases that cleave specific target proteins during apoptotic death (8). We have previously shown that huntingtin is cleaved in apoptotic cells and by recombinant caspase-3 (9), and expression of truncated htt fragments with expanded polyglutamine repeats is known to be toxic to cells (10 -14). These observations led to the development of the toxic fragment hypothesis (15), which postulates that proteolytic cleavage of htt liberates toxic fragments containing the expanded polyglutamine tract that are neurotoxic and that stimulate additional proteolytic activity.Evidence of htt cleavage in HD includes the presence of N-terminal htt fragments in patient brains (16) as well as in yeast artificial chromosome transgenic mice that express fulllength, expanded human htt (17). htt cleavage in the yeast artificial chromosome transgenic mice occurs in the cytoplasm, after which the N-terminal fragments are imported into the nucleus (17).In vitro, htt is cleaved by caspase-3 at two sites yielding N-terminal fragments of 70 and 80 kDa for htt with 15 glutamines and 90 and 100 kDa for htt with 138 glutamines (9, 18). These fragments are also generated when htt is incubated with apoptotic extracts (9, 18) and accumulate from endogenous htt in apoptotic cells (19). Taken together, these results suggest that caspase-3 is likely to contribute to the generation of N-terminal htt fragments.Further support for the toxic fragment hypothesis can be obtained by testing whether preventing the formation of Nterminal htt fragments lessens the toxicity of htt. Here we show abrogation of htt c...

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

“…Cleavage of Htt by caspase-3 yields a 70 kDa (80 kDa; expanded Htt) fragment and 75 kDa (85 kDa; expanded Htt) fragment (sites at D513, D552), while caspase-6 cleavage yields an 80 kDa (90 kDa; expanded Htt) fragment (cleavage site at D586). 36 As shown in Figure 1b,c, caspase-2 is also capable of cleaving Htt to generate a cleavage product at approximately 75 kDa (85 kDa; expanded Htt), which corresponds to cleavage at D552. Comparison of Htt cleavage by caspase-2, -3, and caspase-6 with various caspase-resistant forms of Htt (Figure 1c) demonstrates that caspase-2 cleaves at D552 in Htt3949-138 yielding an 85 kDa cleavage product.…”

“…[34][35][36] We have previously shown that Htt is cleaved in vitro by caspase-3 at amino acids 513 and 552, and by caspase-6 at amino-acid position 586. 36 However, in vivo, we found that Htt is specifically cleaved at a caspase consensus site at amino acid 552 in pyramidal cortical neurons, while the caspase consensus site at amino acid 513 does not appear to be utilized. 31 Since caspase-3 cleaves at both D513 and D552, we further evaluated the specificity of caspases in cleaving Htt.…”

“…28,34,36 The following catalytic mutant caspases, which disable the catalytic cysteine residue, were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene): caspase-2 (C303A), caspase-3 (C163A), caspase-6 (C163A), caspase-7 (C186A), caspase-8 (C360A), and caspase-9 (C287A).…”

Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability

“…Nonpathological length polyglutamine peptides are inherently capable of forming fibrils on their own, albeit slowly (20), yet this behavior generally is not observed in the context of an entire protein. Furthermore, sequences of the regions immediately flanking the polyglutamine tract have been shown to influence the rate of aggregation (52), and some polyglutamine proteins undergo necessary proteolysis prior to aggregation, with the release of a polyglutamine-containing fragment that subsequently forms fibrils (53)(54)(55)(56). Therefore, it is conceivable that domains such as the Josephin domain in ataxin-3 may play a role in maintaining the solubility of the protein, or exert a restraining force such that the conformational shift to the misfolded form is impeded or is energetically less favorable.…”

“…However, upon expansion, the native random coil conformation of the polyglutamine tract becomes a more prominent feature of the overall protein structure, and more importantly, the intrinsic propensity of the polyglutamine tract to adopt an inappropriate conformation may be strengthened, and the counteractive influence of the external regions to this transition may become less effective. Similarly, the disruption of nonpolyglutamine domains and regions under denaturing conditions (24,25) or their removal by cleavage (53)(54)(55)(56) would also result in their loss of ability to prevent the transition to the misfolded conformer.…”