HD is caused by a mutation encoding a polyglutamine expansion at the N-terminus of Htt protein. It has been postulated that proteolytic processing of Htt leads to toxic fragments that drive disease progression, so the enzymes involved are potential therapeutic targets for HD. Previously, mice expressing mHtt that is not cleaved by caspase-6 but is cleaved by caspase-3 were shown to have normal neuronal function and were not susceptible to neurodegeneration (Graham et al., 2006
). We therefore investigated whether the putative protease responsible for cleavage of Htt at Asp-586 was disease-modifying in vivo
. Specifically, we crossed BACHD mice with Casp6−/−
Previously, caspase-6 was suggested to be the key protease that cleaves Htt at aa586 (Graham et al., 2006
), and so a homozygous caspase-6 knockout would be predicted to eliminate production of this Htt fragment. While we found that knocking out caspase-6 reduced expanded full-length Htt levels and overall Htt fragment levels, surprisingly it did not eliminate production of the aa586 Htt cleavage product. Interestingly, the YAC128 C6R mouse that showed full neurological rescue (Graham et al., 2006
) was mutated at both the aa586 and aa589 sites, suggesting that either cleavage site may be involved in the phenotypic rescue. Alternatively, like the secretase pathways where mutation of the amyloid precursor protein site changes activity of the secretases, the rescue of YAC128 C6R could be due to altered protease activity (Golde et al., 2011
). Analysis of a conditional caspase-6 knockout crossed to a HD mouse models is required to eliminate the possibility of compensatory pathways explaining our results. Further experiments will need to be conducted to confirm the cleavage site and key protease involved in Htt fragmentation.
No HD-like phenotypic abnormalities have been reported in the YAC128C6R mouse model (Graham et al., 2006
) where a YAC128 mHtt transgene harboring the caspase-6 motif mutations (aa586 and aa589) was introduced into the mouse by transgenesis. Given the hypothesis that absence of caspase-6 cleavage at the mutated site in the YAC128 Htt protein leads to mice lacking an HD phenotype, we sought to independently investigate the effects of caspase-6 ablation on the general health, motor, and MRI phenotypes of the BACHD mouse model. BACHD Casp6−/−
mice showed significant improvement in rotarod motor performance compared to BACHD mice. The reduction in both soluble and insoluble mHtt aggregates may be responsible for this motor improvement, since reduction in expanded Htt levels has been shown to have a protective effect in HD (Ravikumar et al., 2002
; Sanchez et al., 2003
; Ravikumar et al., 2004
). However, it should be noted that recent studies show that body weight can in part modulate rotarod performance in the BACHD line (unpublished data), making it possible that the improvement in rotarod performance in BACHD Casp6−/−
mice is at least partly due to their reduced body weight. Interestingly, the reduced body weight in BACHD Casp6−/−
and BACHD Casp6+/−
animals may be a consequence of the unexpected reduction of full-length expanded Htt since it is known that full-length Htt levels in YAC128 mice can modulate body weight (Van Raamsdonk et al., 2006
We have previously found that body weight also modulates the open field performance of mice, with smaller mice tending to present higher locomotor and rearing activity (unpublished data). Consequently the lack of effects of the caspase-6 knockout on locomotor and rearing activity exhibited by experimental mice in the open field task might be considered surprising given that they are significantly smaller than their WT counterparts. It is possible, however, that the knockout of caspase-6, independent of the BACHD genotype, induces a reduction of locomotor activity that is then masked by weight-related increases in activity; this possibility should be further investigated in the Casp6−/− mouse.
Our data do not support a neuroprotective effect of eliminating caspase-6 activity on mHTT-mediated whole brain and striatal atrophy, as measured in the BACHD mouse. Although these atrophies are modest in the BACHD mice we have no evidence that caspase-6 knock out prevents such changes.
A surprising finding of this study is the decrease in expanded full-length mHtt and mHtt fragments. We present preliminary evidence that soluble mHtt lowering and loss of mHtt aggregates may be modulated through enhanced protein clearance pathways in BACHD Casp6−/−
mice. Interestingly, it has recently been shown that beclin, p62 and hAtg3 - each involved in autophagy - are substrates for caspase-6 cleavage (Norman et al., 2010
). This connection between caspase-6 and protein clearance pathways led us to hypothesize that the Casp6−/−
mice might exhibit altered levels of autophagy or ubiquitin proteasome system (UPS) activation. Our immunohistochemical analyses of BACHD Casp6−/−
brain tissue suggests increased LC3 expression, with a redistribution in cortical neurons, as well as a decrease in p62 is consistent with the hypothesis that caspase-6 may be modulating autophagic clearance of mHtt. Furthermore, in cortical neurons of BACHD Casp6−/−
mice we found evidence for increased association of p62 and K444-acetylated Htt relative to BACHD mice; the latter is a substrate for autophagy-mediated clearance (Jeong et al., 2009
), and our results suggest it is altered in the homozygote Casp6−/−
mice. Further work is required for a detailed understanding of the specific mechanisms engaged in the BACHD Casp6−/−
mice that lead to lower levels of soluble mHtt and insoluble inclusion bodies. Autophagy would seem a viable candidate but our data showing a redistribution of ubiquitin staining and co-localization of Htt and ubiquitin in BAC Casp6−/−
mouse brain raises the possibility that the UPS may be involved.
In summary, we show that elimination of caspase-6 protein and activity in the BACHD mouse model does not prevent the production of a 586 amino acid Htt proteolytic fragment in the brain. This data suggests that generation of this fragment in vivo
is not due exclusively to caspase-6 activity and raises serious concerns about pursuing caspase-6 as a therapeutic target for HD in the context of the proteolytic fragment hypothesis. Studies using the HdhQ150 knockin model (Lin et al., 2001
) crossed to the Casp6−/−
mice saw no change in the general pattern of Htt fragmentation or level of the Htt586 product further supporting this conclusion (G. P. Bates, personal communication). Further investigation into cleavage of Htt at the aa586 and aa589 sites is required to verify the key cleavage site, identify the protease responsible, and explore whether protease inhibition is a viable therapeutic strategy. An siRNA screen monitoring Htt586 or Htt589 cleavage levels as the endpoint may be an unbiased way to identify other potential proteases involved in this pathway. Future work directed at dissecting the role of caspase-6 in HD may require the use of conditional knockdown strategies to resolve the contributions of clearance pathways and Htt proteolysis.