In this study we have generated a novel transgenic mouse model of Huntington’s disease using the putative caspase 6 derived Htt fragment “N586” with an expansion length of 82 glutamines. Similar to our previous N171-82Q model, the N586-82Q model has a robust and progressive behavioral phenotype, with early brain atrophy and early mortality. There is accumulation of Htt aggregates, mostly consisting of the N586 polypeptide. In addition, we see striking evidence for time-dependent proteolysis of the N586 polypeptide into smaller soluble N-terminal fragments, which are highly enriched in the nucleus ().
Summary of N586 82Q proteolysis and aggregation pathway
Initial characterization of the N586 mouse model phenotype suggests that it has an intermediate phenotype relative to the previous fragment models and full-length models. The phenotype is quite similar to that of both the N171-82Q and R6/2 models: there is progressive weight loss and an end stage which includes rigidity, hypoactivity, and poor grooming. A recent study showed that expression of mutant Htt in the hypothamus causes metabolic abnormalities and induces obesity in mice. This effect was independent of fragment size (Hult et al., 2011
). Therefore, if N586-82Q were to be expressed only in the hypothalamus, a similar outcome might be expected. Like the N-terminal fragments expressed in the N171-82Q and R6/2 mouse models, N586-82Q also induces premature death. We have not yet characterized the behavioral time course of this model in detail, but the studies done so far indicate that initial hyperactivity progresses to hypoactivity at later stages. This is somewhat reminiscent of human HD, in that hyperkinetic movement disorder tends to predominate early in the course, but hypokinetic movement supervenes later in the course. In addition to motor disturbances, we show abnormalities of cognitive and emotional behavior. This is also comparable to human HD, which has motor, cognitive and emotional components.
The N586-82Q fragment is intermediate in size compared to full length Htt and the fragments used to create either the N171-82Q or R6/2 mice. The N586-82Q fragment appears to induce a phenotype which progresses faster than full length models, but slower than models made with shorter fragments. The N586-82Q mice express much greater amounts of protein than the N171-82Q mice. Since the expression of both proteins, N586 and N171 are under control of the same promoter and have the same length polyglutamine length it appears that it is not the amount of protein expressed which controls the severity of the ensuing phenotype, but rather the fragment size.
The comparison of Htt protein levels between the YAC128 and N586-82Q mice is slightly more complex. Htt expression in the YAC128 mice is under the control of human Htt promoter and the polyglutamine tract is considerably longer. Anti-Htt 1–82 revealed a somewhat higher expression pattern in the N586-82Q. The 1C2 antibody reacts more strongly with longer repeat lengths, and shows very similar reactivity to Htt in the two models. Based on these findings, the accelerated phenotype of the N586-82Q mice compared to full-length models may not be due only to higher protein expression but rather due to the expression of a shorter polyglutamine containing polypeptide. Although, it is reasonable to assume that the different promoters driving Htt expression in these mice might introduce variations, our data suggest that the N586-82Q fragment being an intermediate size induces an intermediate phenotype.
Brain MRI studies show substantial brain atrophy in this model, which can be described as degenerative rather than developmental, since younger mice have regional normal brain volumes. Atrophy is relatively widespread, which is comparable to previously described fragment models such as the R6/2 and the N171-82Q model. In human HD there is widespread brain atrophy at late stages, with brains of late-stage HD patients showing overall atrophy comparable to those of late stage Alzheimer’s patients, and more widespread atrophy in patients with long repeat expansions. By contrast full length models may have more regionally selective atrophy.
A striking feature of our model is the evidence for further proteolytic cleavage of the N586 polypeptide into smaller fragments. The fastest migrating fragment is the most abundant, and appears to accumulate over time. While we have not determined the exact cleavage sites, the epitope mapping indicates that the predominant fragment is similar to what we have described in cell culture studies as “cp-1” (Ratovitski et al., 2007
; Ratovitski et al., 2009
). A recent study (Landles et al., 2010
) of proteolytic cleavage in the 140Q knock-in mouse model shows cleavage of Htt into a number of fragments. One of these fragments was proposed on the basis of antibody reactivity to be exon 1. The approximate migration of the fragment in our model is comparable to both the exon 1 and cp-1 fragments. Since N586-82Q predominated in the cytoplasmic fraction, where low amounts of soluble N-terminal fragments were found, we hypothesize that N586-82Q is cleaved in the cytoplasm, and that the fragments generated migrate to the nucleus. It is striking that the nuclear fraction consists almost entirely of N-terminal fragments rather than the N586 polypeptide itself, suggesting that the N586 polypeptide, if formed from full length Htt in the disease process, may be a transient intermediate.
A previous study found that caspase 6 cleavage of full length Htt could take place inside the nucleus. However, overexpression of N586 tends to result in cytoplasmic localization. In our mouse model, N586-82Q is overexpressed, and is largely located in the cytoplasm. It is possible that the N586-82Q polypeptide enters the nucleus first and is then degraded into smaller N-terminal fragments. However, this may be less likely, given that N586-82Q was found only in the cytoplasm even at a younger age. The cell biology of Htt nuclear import and export is still not completely understood (Trushina et al., 2003
; Xia et al., 2003
; Cornett et al., 2005
; Havel et al., 2009
), but since many studies have indicated that nuclear N-terminal Htt fragments are more toxic than those in the cytoplasm, we hypothesize that their accumulation in this model is important for toxicity.
The appearance of the shortest fragments in the N586-82Q mice appears to correlate with the development of the behavioral phenotype. This is consistent with the idea that the small cp-1 like fragment has pathogenic relevance. Interestingly, the YAC128 model does not accumulate as much of this fragment as the N586-82Q model. Since we believe the cp-1 fragment is a toxic fragment, this might help explain the rate of disease progression in the two models--the N586-82Q mice accumulate more of the cp-1 like fragment and therefore develop a phenotype sooner than the YAC128.
In parallel with our project,Tebbemkamp et al developed a mouse model expressing N586-82Q under the control of the prion promoter, as in our model, but also expressing eGFP under the control of the keratin 14 promoter (K14-eGFP) (Tebbenkamp et al., 2011
). The single line of mice reported showed profound ataxia, attributed to cerebellar granule cell degeneration observed in that line. This feature was not present in our N586-82Q model. Furthermore, the authors did not observe the same proteolysis pattern found in our mice. The cytoplasmic aggregates in Tebbencamp et al mice are apparently composed largely of small fragments and not the N586-82Q polypeptide itself. By contrast, the Htt aggregates in the cytoplasm of our model are made up mostly by the N586-82Q polypeptide. The authors conclude that the ataxia phenotype in their model is due to cerebellar granule cell degeneration, possibly because ‘enhancer elements in the K14 promoter affect the temporal or spatial pattern of htt N586-82Q expression in some manner that modifies disease phenotype’. Such marked cerebellar granule cell degeneration is not a feature of human HD. Therefore, we believe the N586-82Q model reported here to be more relevant for HD pathogenesis.
In conclusion, we have generated a new HD mouse model which demonstrates that the putative caspase 6 derived Htt polypeptide of 586 amino acids is a toxic fragment (). This model has some favorable features for future studies. It expresses a postulated physiologically relevant fragment. This fragment is considerably longer than those in previous fragment models, and thus will be more suitable for studying post-translational modifications. There are many known post-translational modification sites in this region, including a number of residues in the first 17 amino acids (Gu et al., 2009
; Tam et al., 2009
) the akt phosphorylation site (S421) (Humbert et al., 2002
), the acetylatation site (K444) (Jeong et al., 2009
), and the lipid modification site (C214) (Yanai et al., 2006
). In addition there are likely to be other sites as well (unpublished data). We show striking evidence that further proteolytic cleavage of Htt into short soluble nuclear fragments may contribute to pathogenesis. Identification of the exact sites of cleavage may provide important insight into HD biology. Behavioral abnormalities in this model begin by approximately four months of age, suggesting that it may be suitable for studies of the “presymptomatic” or “prodromal” period (Aylward et al., 2010
; Ross and Tabrizi, 2011
; Tabrizi et al., 2011
). The model has a robust and progressive behavioral and neuropathological phenotype, with death at approximately one year, making it suitable for preclinical therapeutic studies.