Abstract
BackgroundHuntington’s disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ) stretch within Huntingtin (htt), the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR) in modulating HD mouse model pathogenesis is currently unknown.ResultsWe describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh) encoding N-terminal hemaglutinin (HA) or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt) and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt). Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q) htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis.ConclusionIn mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.
Highlights
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine stretch within Huntingtin, the protein product of the HD gene
The 140Q stretch was derived from our CAG140 targeting construct [32], and contains the human proline-rich region (PRR) that is adjacent to the polyQ stretch
To determine if astrocytosis in Hdh140Q/3xFlag20Q mice is altered in comparison to that observed in Hdh140Q/3xFlag7Q mice, we examined glial fibrillary acidic protein (GFAP) expression by immunohistochemistry in the striatum of 24 month-old wild-type, Figure 7 The mean size of htt inclusions in the Hdh140Q/3xFlag20Q striatum is significantly larger than that in the Hdh140Q/3xFlag7Q striatum at 2 years of age. (A) Confocal images of the Hdh140Q/3xFlag7Q and Hdh140Q/3xFlag20Q striatum at 24 months of age
Summary
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ) stretch within Huntingtin (htt), the protein product of the HD gene. Despite a correlation between the appearance of htt aggregates and behavioral deficits in the majority of HD mouse models, the role of these inclusions in the mechanism of HD pathogenesis is still uncertain, as the results from in vitro experiments and some HD mouse models have suggested that large visible mutant htt inclusions are neuroprotective [7,8,9,10,11] Such aggregates have the ability to recruit toxic soluble fragments or oligomers of mutant Htt, but they can sequester other polyQ-containing proteins, including wild-type htt [12,13,14,15]. Despite evidence from in vitro experiments and a Drosophila model supporting the hypothesis that truncated N-terminal fragments of mutant Htt can sequester wildtype htt fragments in aggregates, evidence for the sequestration of wild-type htt by mutant htt in mouse models is lacking
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