Transgenic Mouse Model Expressing the Caspase 6 Fragment of Mutant Huntingtin

Division of Neurobiology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.75). 01/2012; 32(1):183-93. DOI: 10.1523/JNEUROSCI.1305-11.2012
Source: PubMed

ABSTRACT Huntington's disease (HD) is caused by a polyglutamine expansion in the Huntingtin (Htt) protein. Proteolytic cleavage of Htt into toxic N-terminal fragments is believed to be a key aspect of pathogenesis. The best characterized putative cleavage event is at amino acid 586, hypothesized to be mediated by caspase 6. A corollary of the caspase 6 cleavage hypothesis is that the caspase 6 fragment should be a toxic fragment. To test this hypothesis, and further characterize the role of this fragment, we have generated transgenic mice expressing the N-terminal 586 aa of Htt with a polyglutamine repeat length of 82 (N586-82Q), under the control of the prion promoter. N586-82Q mice show a clear progressive rotarod deficit by 4 months of age, and are hyperactive starting at 5 months, later changing to hypoactivity before early mortality. MRI studies reveal widespread brain atrophy, and histologic studies demonstrate an abundance of Htt aggregates, mostly cytoplasmic, which are predominantly composed of the N586-82Q polypeptide. Smaller soluble N-terminal fragments appear to accumulate over time, peaking at 4 months, and are predominantly found in the nuclear fraction. This model appears to have a phenotype more severe than current full-length Htt models, but less severe than HD mouse models expressing shorter Htt fragments. These studies suggest that the caspase 6 fragment may be a transient intermediate, that fragment size is a factor contributing to the rate of disease progression, and that short soluble nuclear fragments may be most relevant to pathogenesis.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites.
    PLoS ONE 02/2014; 9(2):e88284. DOI:10.1371/journal.pone.0088284 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A long-term goal of modeling Huntington's disease (HD) is to recapitulate the cardinal features of the disease in mice that express both mutant and wild-type (WT) huntingtin (Htt), as HD commonly manifests as a heterozygous condition in humans, and loss of WT Htt is associated with loss-of-function. In a new heterozygous Q175 knock in (KI) mouse model, we performed an extensive evaluation of motor and cognitive functional deficits, neuropathological and biochemical changes, and levels of proteins involved in synaptic function, the cytoskeleton, and axonal transport, at 1-16 months of age. Motor deficits were apparent at 6-months of age in Q175 KI mice and at that time, post-mortem striatal GABA levels were elevated and mutant Htt inclusions were present throughout the brain. From 6 months of age, levels of proteins associated with synaptic function, including SNAP-25, Rab3A and PSD-95, and with axonal transport and microtubules, including KIF3A, dynein and dynactin, were altered in the striatum, motor cortex, pre-frontal cortex and hippocampus of Q175 KI mice, compared to wild-type levels. At 12-16 months of age, Q175 KI mice displayed motor and cognitive deficits, which were paralleled at post-mortem by striatal atrophy, cortical thinning, degeneration of medium spiny neurons, dense mutant Htt inclusion formation, decreased striatal dopamine levels and loss of striatal BDNF. Data from this study indicate that the heterozygous Q175 KI mouse represents a realistic model for HD and also provides new insights into the specific and progressive synaptic, cytoskeletal and axonal transport protein abnormalities that may accompany the disease.
    Human Molecular Genetics 04/2014; DOI:10.1093/hmg/ddu166 · 6.68 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Huntington's disease (HD) is a progressive autosomal dominant disorder, caused by a CAG repeat expansion in the HTT gene, which results in expansion of a polyglutamine stretch at the N-terminal end of the huntingtin protein. Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis and it is generally accepted that N-terminal huntingtin fragments are more toxic than full-length protein. Important cleavage sites are encoded by exon 12 of HTT. Here we report proof of concept using antisense oligonucleotides to induce skipping of exon 12 in huntingtin pre-mRNA, thereby preventing the formation of a 586 amino acid N-terminal huntingtin fragment implicated in HD toxicity. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein. Cleavage assays showed reduced 586 amino acid N-terminal huntingtin fragments in the treated samples. In vivo studies revealed exon skipping after a single injection of antisense oligonucleotides in the mouse striatum. Recent advances to inhibit the formation of mutant huntingtin using oligonucleotides seem promising therapeutic strategies for HD. Nevertheless, huntingtin is an essential protein and total removal has been shown to result in progressive neurodegeneration in vivo. Our proof of concept shows a completely novel approach to reduce mutant huntingtin toxicity not by reducing its expressing levels, but by modifying the huntingtin protein.
    12/2013; 24(1). DOI:10.1089/nat.2013.0452

Full-text (2 Sources)

Available from
May 30, 2014