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, Vismodegib concentration 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 analyses 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. Prior to this study, the expression of an expanded antisense CAG-containing transcript or a novel polyQ protein had not been demonstrated by using postmortem patient brain tissues (Holmes et al., 2001 and 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 selleck compound 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 wild-type 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 with antibodies that provide signal strength in western blots depending on the polyQ length (e.g., 3B5H10).

Because the brain regional and subcellular Adenosine 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 is also likely to be similar. Future studies with 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).