We have generated and characterized the first BAC transgenic mouse models of an HD-like disorder, HDL2. BAC-HDL2 mice recapitulate several key phenotypes found in HDL2 patients, including age-dependent motor deficits and selective forebrain atrophy. They also capture two molecular pathogenic hallmarks of HDL2: the progressive accumulation of ubiquitin-positive NIs, and the presence of CUG containing RNA foci that are not co-localized with NIs. Importantly, this model reproduces the brain region specific distribution of NIs seen in the patients, suggesting that the mechanism underlying the pathogenesis of NIs is likely reproduced in this mouse model. Furthermore, the disease phenotypes are not present in control BAC mice without the CTG/CAG repeat expansion.
A significant finding of our study is the discovery of a novel molecular mechanism leading to polyQ pathogenesis in BAC-HDL2 mice (see schematics in ). Using our series of novel mouse models, we first demonstrated the expression of an expanded CAG-containing transcript from the strand antisense to JPH3, with its expression driven by a novel promoter. Second, immunohistochemistry and Western blot analyses demonstrated that this transcript is expressed as a protein containing an expanded polyQ tract. Third, we used two antibodies relatively specific for polyQ to demonstrate the accumulation of NIs containing polyQ in two independent lines of BAC-HDL2 mice and found the spatial distribution of NI accumulation to be strikingly similar to that found in HDL2 patients. Fourth, we used two control BAC mouse lines with normal CTG/CAG repeats to demonstrate that disease pathogenesis in BAC-HDL2 mice, including NI formation, is dependent on the CTG/CAG repeat expansion. Finally, by genetic silencing of the JPH3 sense strand in BAC-HDL2-STOP mice, preventing expression of CUG-containing transcripts, we provided definitive genetic evidence that the expression of the HDL2-CAG transcript alone can lead to the formation of polyQ-containing NIs and manifestation of motor deficits. Taken together, our analysis of the series of HDL2 mouse models provide an important mechanistic insight that the expression of a novel expanded polyQ protein could play a critical role in HDL2 pathogenesis in vivo.
Figure 8 A schematic model for novel pathogenic mechanisms revealed by HDL2 mouse model in which a novel antisense expanded CAG transcript is mediating polyQ pathogenesis in vivo. A novel expanded CAG repeat (HDL2-CAG) antisense to the JPH3 sense strand is transcribed (more ...)
Prior to this study, the expression of an expanded antisense CAG-containing transcript or a novel polyQ protein had not been demonstrated using postmortem patient brain tissues (Holmes et al., 2001
; Margolis et al., 2005
). There are several possible explanations to account for the more sensitive detection of the CAG transcript and polyQ expanded protein in HDL2 mice than in patients. First, unlike the postmortem brain tissues, the HDL2 mouse brains used for the studies do not exhibit robust neurodegeneration that may lead to the loss of neurons expressing mutant CAG transcripts at high levels. Second, the sequence difference between the human mutant JPH3 transgene and murine wildtype locus in the same HDL2 mouse permits the easier detection of the mutant antisense transcripts. Third, the much longer polyQ repeat in the BAC-HDL2 mouse model (120Q, as compared to 40–50Q in patients) also permits more sensitive detection of the small amount of soluble mutant polyQ protein using antibodies that provide signal strength in Western blots depending on the polyQ length (e.g.
3B5H10). Since the brain regional and subcellular distribution of the NIs are remarkably similar between HDL2 mice and patients, it would strongly argue that the molecular pathogenesis for such NIs in HDL2 mice and patients are also likely to be similar. Future studies using additional patient samples and more sensitive detection methods may be needed to demonstrate HDL2-CAG transcript and protein in the patient brains.
Although our study is focused on the mechanistic investigation of the novel antisense CAG repeat transcript and its polyQ containing protein product, our study does not exclude a contribution of other potential mechanisms to aspects of HDL2 pathogenesis, such as partial loss of JPH3 function or CUG repeat RNA gain-of-function toxicity (Rudnicki et al., 2007
). Despite the fact that our study was not designed to address these alternative mechanisms, our models do support a potential role of CUG RNA in disease pathogenesis, since BAC-HDL2 mice accumulate CUG RNA foci that can sequester Mbnl1 hence mimicking those in the patients. Importantly, the CUG RNA foci and NIs in both HDL2 mice and patients appear to be distinct entities, consistent with the interpretation that independent pathogenic mechanisms lead to their formation (ibid
; Supplemental Figure S2
, Panel B). Since expanded CUG repeat RNA is clearly pathogenic in DM1 via an RNA gain-of-function mechanism (Mankodi et al., 2001
), in part via sequestration and depletion of MBNL1 (Kanadia et al., 2003
), future mouse genetic studies are needed to address whether the expression of expanded CUG repeat transcripts also could mediate CUG repeat RNA toxicities in vivo
An overarching goal of this study was to shed light on potential common pathogenic mechanisms shared between HD and HD-like disorders. By the development and analysis of the first BAC mouse genetic model for an HD-like disorder, we have already gained some initial insights toward this important objective. First, our cumulative analysis of these models suggests that expanded polyQ-mediated pathogenesis may be a shared pathogenic mechanism between HD and HDL2. The finding that a mutant polyQ protein may contribute to HDL2 pathogenesis in BAC-HDL2 mice is consistent with the presence of NIs that stain with both 1C2 and 3B5H10 antibodies in HDL2 brain, and the comparable pathogenic CAG repeat threshold for HDL2 and HD (about 40 triplets). It is striking that this threshold is similar to other polyglutamine diseases, but is much shorter than the threshold for most of the non-polyglutamine repeat expansion disorders, most prominently DM1. Thus, our study provides experimental evidence to suggest that therapeutics that could ameliorate expanded polyQ toxicity could benefit both HD and HDL2.
Another potentially shared pathogenic mechanism between HD and HDL2 is transcriptional dysregulation mediated by sequestration and/or functional interference of CBP. Prior studies have demonstrated that mutant huntingtin N-terminal fragments can bind to and/or sequester CBP into aggregates leading to changes in CBP mediated transcription (Kazantsev et al., 1999
; Nucifora et al., 2001
; Steffan et al., 2000
). Although physical depletion of CBP is not consistently observed in all HD mouse models (Yu et al., 2002
), functional interference of CBP has been observed in HD as well as other polyQ disorders. Indirect restoration of CBP function via histone deacetylase (HDAC) inhibition has been shown to be beneficial in several animal models of HD (Steffan et al., 2001
) and in other polyQ disorders such as SBMA (McCampbell et al., 2000
; Taylor et al., 2003
). Our analysis of BAC-HDL2 mice provides evidence that expression of BNDF is significantly reduced in the mutant cortex at 15 months of age, and our in vivo
ChIP analysis and primary neuron study reveal that mutant HDL2-CAG can selective interfere with CBP co-activator function at BDNF promoter IV (Hong et al., 2008
). Together, our study supports the notion that mutant HDL2-CAG protein can perturb CBP-mediated transcription, in part but not necessarily exclusively, through the sequestration of CBP into NIs. Our current study does not rule out the possibility that mutant HDL2-CAG protein may also disrupt the function of other critical nuclear transcription factors such as TBP (Rudnicki et al., 2008
). Additional gene expression and epigenetic profiling, along with functional manipulation of these molecular pathways in HDL2 mice, will be necessary to critically evaluate their contribution in disease pathogenesis.
Finally, our study reveals the complexity of disease pathogenesis mediated by trinucleotide repeat expansion. Together with SCA8 (Moseley et al., 2006
), our models provide another compelling example in which bi-directional transcription across an expanded CTG/CAG repeat leads to the expression of an antisense CAG transcript and previously unrecognized polyQ protein toxicity. Since the predicted HDL2-CAG protein has no known homology to any other protein in the human proteome beyond the polyQ stretch (data not shown), the function of this novel transcript and the small protein it encodes remains to be explored. Given the recent discovery that antisense transcription is nearly ubiquitous throughout the mammalian genome (Katayama et al., 2005
), our study highlights the importance of examining antisense repeat-containing transcripts and their ORFs in the pathogenesis of other brain disorders.