Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis

Abstract

A polyglutamine expansion located in the N terminus of huntingtin (N-htt) causes Huntington's disease (HD). How the mutation causes cell death is unknown. Several recent observations implicate altered huntingtin (htt) processing in the pathogenesis of HD. In the HD brain, mutant N-htt fragments aggregate in the nucleus and cytoplasm (1); the expression of mutant N-htt fragments in vitro causes cell death (2, 3). These findings suggest that proteolysis in the N-terminal region of htt may be important in HD pathogenesis. Furthermore, htt cleavage by caspase 3 could contribute to neurodegeneration in HD. Htt can serve as a substrate for caspase activity. Two proximate caspase 3-sensitive sites and one caspase 6-sensitive site are distal to the polyglutamine tract at aspartate residues 513, 552, and 589, respectively, in the wild-type (wt) protein (4-6). Caspase 3-cleaved N-htt products have been observed in vitro after exogenous expression of human htt in HEK 293 cells (4) and clonal striatal neurons (7). Treatment with the broad acting caspase inhibitor Z-VAD-FMK attenuates caspase 3 cleavage of htt and increases cell survival (6, 7). Enhanced immunoreactivity for caspases has been reported in HD striatal neurons compared with control brain, § supporting the involvement of caspase activation in HD pathogenesis. However, there is no evidence for htt proteolysis by caspases in the brain. No non-caspase proteases have been identified that produce a limited proteolysis of htt. Thus, despite the presence of N-htt fragments in adult and juvenile HD brain (1), the proteolytic pathway involved in the production of mutant N-htt fragments in the brain is unknown. That caspase 3 or other proteases cleave the N terminus of mutant htt in the HD brain would provide strong support that N-terminal htt proteolysis is a critical factor in HD pathogenesis.Here, we demonstrate that caspase 3-cleaved N-htt fragments occur in the control and HD brain and are similar in size to fragments produced in vitro after exogenous expression of human wt and mutant htt in mouse clonal striatal cells (7,8). The wt and mutant caspase 3-cleaved N-htt fragments in brain varied in size with polyglutamine length and were preferentially enriched in membrane fractions. Partial proteolysis of the caspase 3-cleaved N-htt fragments by calpain produced smaller Nterminal fragments. We speculate that caspase 3 cleavage regulates the proteolysis of wt and mutant htt in the brain and increases the association of N-terminal htt with membranes. Calpain-induced proteolysis of the caspase 3-cleaved mutant N-htt fragment may lead to the formation of mutant N-htt fragments that can aggregate and form inclusions. Materials and MethodsCell Culture and Transfections. The culturing and transfection of mouse clonal striatal cells (X57 cells) have been described in our recent publications (7,8,9). MCF-7 cells were grown according to the suppliers' recommendations [American Tissue Culture Collection (ATCC)]. DNA was introduced by using Superfect Transfection Reagent (Qiagen, ...

Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease

“…The Htt constructs were engineered to include the first 548 aa of the human Htt protein, which include and extend well beyond the 81-aa product encoded by the first exon of the gene. The 548-aa fragment is truncated close to the site of cleavage by caspase-3, thought to be a crucial step in the generation of aggregate-forming Htt fragments (17,18). This region also encompasses the highest stretch of homology between the Drosophila and human Htt proteins (Fig.…”

Viral delivery of glial cell line-derived neurotrophic factor improves behavior and protects striatal neurons in a mouse model of Huntington’s disease

“…It has been suggested that mutant huntingtin is cleaved and translocates into the nucleus, where it activates apoptosis (43). Additionally, it has been demonstrated that the lack of normal huntingtin in HD patients and transgenic mice leads to a decrease in the transcription of trophic factors, such as BDNF (44).…”

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.…”