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: 6.34). 01/2012; 32(1):183-93. DOI: 10.1523/JNEUROSCI.1305-11.2012
Source: PubMed


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.

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Available from: Rona K Graham, Jan 15, 2014

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Article: Transgenic Mouse Model Expressing the Caspase 6 Fragment of Mutant Huntingtin

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    • "Neurodegeneration in HD affects mostly the GABAergic medium spiny neurons of the striatum (Albin et al., 1990; Vonsattel et al., 1985). Proteolysis of mutant HTT (mHTT) is implicated in the pathogenesis of HD (DiFiglia et al., 1997; Gafni et al., 2004; Graham et al., 2006a, 2010; Kim et al., 2001; Maglione et al., 2006; Waldron-Roby et al., 2012; Warby et al., 2008; Wellington et al., 2002). Of particular interest is the site at amino acid (aa) 586 containing a caspase-6 (Casp6) recognition motif (Graham et al., 2006a). "
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    ABSTRACT: Huntington Disease (HD) is a progressive neurodegenerative disease caused by an elongated CAG repeat in the huntingtin (HTT) gene that encodes a polyglutamine tract in the HTT protein. Proteolysis of the mutant HIT protein (mHTT) has been detected in human and murine HD brains and is implicated in the pathogenesis of HD. Of particular importance is the site at amino acid (aa) 586 that contains a caspase-6 (Casp6) recognition motif. Activation of Casp6 occurs presymptomatically in human HD patients and the inhibition of mHTT proteolysis at aa586 in the YAC128 mouse model results in the full rescue of HD-like phenotypes. Surprisingly, Casp6 ablation in two different HD mouse models did not completely prevent the generation of this fragment, and therapeutic benefits were limited, questioning the role of Casp6 in the disease. We have evaluated the impact of the loss of Casp6 in the YAC128 mouse model of HD. Levels of the mHTT-586 fragment are reduced but not absent in the absence of Casp6 and we identify caspase 8 as an alternate enzyme that can generate this fragment. In vivo, the ablation of Casp6 results in a partial rescue of body weight gain, normalized IGF-1 levels, a reversal of the depression-like phenotype and decreased HIT levels. In the YAC128/Casp6-/- striatum there is a concomitant reduction in p62 levels, a marker of autophagic activity, suggesting increased autophagic clearance. These results implicate the HTT-586 fragment as a key contributor to certain features of HD, irrespective of the enzyme involved in its generation.
    Neurobiology of Disease 01/2015; 76. DOI:10.1016/j.nbd.2014.12.030 · 5.08 Impact Factor
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    • "A cleavage by caspase 6 at position 586 is believed to be one of the first steps of the toxic proteolysis of Htt [23]. Transgenic mouse models expressing the caspase 6 fragment or other shorter fragments generally have more striking and robust phenotypes than transgenic mouse models expressing full-length Htt [20], [24]–[27]. Downstream steps in the pathogenic process likely include nuclear localization and accumulation resulting in alterations of transcription, abnormal proteostasis, and interference with metabolic and mitochondrial function. "
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    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 · 3.23 Impact Factor
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    ABSTRACT: Huntington’s disease (HD) is one of the most common dominantly-inherited neurodegenerative disorders and is caused by a CAG repeat expansion in the huntingtin gene. HD is characterized by selective degeneration of subpopulations of neurons in the brain, however the precise underlying mechanisms how a ubiquitously expressed disease protein could target specific types of neurons for degeneration remains a critical, yet unanswered question for HD and other major neurodegenerative disorders. In this review, we describe the expanding view of selective neuronal vulnerability in HD, based on recent neuropathological and neuroimaging studies. We will also summarize the systematic effort to define the cell types in which mutant Huntingtin expression is critical for pathogenesis of vulnerable neurons in the striatum and cortex. Finally, we will describe selected, emerging molecular mechanisms that are implicated in selective disease processes in HD. Together, the field has begun to appreciate the distinct molecular pathogenic roles of mutant huntingtin in different cell types that may contribute to the selective neuronal vulnerability, with dissection of such mechanisms likely to yield novel molecular targets for HD therapy.
    10/2012; 7(5). DOI:10.1007/s11515-012-1246-7
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