Caspase-6 Activity in a BACHD Mouse Modulates Steady-State Levels of Mutant Huntingtin Protein But Is Not Necessary for Production of a 586 Amino Acid Proteolytic Fragment

Abstract

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.

Integration-independent Transgenic Huntington Disease Fragment Mouse Models Reveal Distinct Phenotypes and Life Span in Vivo

“…However, subsequent work in BAC Huntington disease mice suggested that caspase-6 knock out modified disease progression through alteration in HTT clearance pathways rather than blocking proteolysis of mutant HTT. These results may reflect compensatory pathways evoked by caspase-6 knock out during development (7). By contrast knock of caspase-2 was shown to reduce behavioral deficits such as performance on the rotarod and forced swim test in YAC128 mice but failed to prevent the reductions in brain weight and changes in volume of subregions of the brain associated with HTT pathology in YAC128 mice (9).…”

“…We focused on comparison of the N552-Q148 and N586-Q148 transgenic mice because although the observed differences in motor, life span, and behavioral phenotypes are distinct, they differ in length by only 34 amino acids and because they represent caspase cleavage products that have been implicated as highly significant in HD disease progression and pathology (5,7,9,12). To explore this hypothesis, interaction studies used two approaches, coIP of full-length HTT (FL-HTT) and two-dimensional BN-PAGE/SDS-PAGE.…”

“…Contributing to the toxicity of mutant HTT protein is the production of protein fragments (5,6). These fragments begin to accumulate and aggregate in neurons during aging and may be necessary to lead to the neurodegeneration that causes HD (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

Partial rescue of some features of Huntington Disease in the genetic absence of caspase-6 in YAC128 mice

“…Constitutive ablation of Casp6 in the BACHD mouse model results in a significant reduction in mHTT aggregates and full-length mHTT levels, likely due to an upregulation of autophagy (Gafni et al, 2012). Additionally, improved performance on the rotarod was shown for BACHD/ Casp6−/− mice (Gafni et al, 2012).…”

“…Additionally, improved performance on the rotarod was shown for BACHD/ Casp6−/− mice (Gafni et al, 2012). Importantly, the mHTT-586 fragment is still present in BACHD/Casp6−/− mice, albeit at reduced levels by immunohistochemical analysis (Gafni et al, 2012).…”