Progressive phenotype and nuclear accumulation of an amino-terminal cleavage fragment in a transgenic mouse model with inducible expression of full-length mutant huntingtin
Introduction
Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the HD gene, coding for polyglutamine in the huntingtin protein (Htt) (Brinkman et al., 1997, Duyao et al., 1993, Huntington's Disease Collaborative Research Group, 1993, Ross et al., 1997, Stine et al., 1993). Onset is usually in mid-adult life but can range from childhood to old age, depending on the length of the CAG repeat expansion. The threshold for the disease is 36 repeats or above, and longer expanded repeats result in earlier onset. The clinical features of HD include movement disorder, cognitive disorder, and behavioral changes, progressively worsening over time, and leading to death. The movement disorder consists of involuntary movements, such as chorea and dystonia, and also progressive incoordination, bradykinesia, and rigidity. Weight loss is often a feature of the phenotype (Djousse et al., 2002). Neuropathologically, HD is characterized by selective neuronal degeneration in the caudate and putamen, as well as by overall brain atrophy (Vonsattel et al., 1985). A pathological hallmark of the disease consists of intranuclear inclusion bodies, which can be labeled with antibodies to the Htt protein, to expanded polyglutamine repeats, or to ubiquitin (Davies et al., 1999).
HD is one of several neurodegenerative disorders caused by expanding polyglutamine repeats. All of the disorders involve neuronal degeneration in selective regions of the brain, distinct in each disease, but with considerable overlap (Ross, 1995). The disease gene products show no homology except for the polyglutamine expansions, leading to the hypothesis that expanded polyglutamine is directly toxic to neurons. While the mechanisms of cell death are incompletely understood, they likely involve largely gain of function mechanisms, mediated in part by translocation of the mutant polyglutamine stretch into the cell nucleus and alterations of gene transcription (Cha, 2000, Ross, 2002, Sugars and Rubinsztein, 2003). Of the polyglutamine diseases, dentatorubral and pallidoluysian atrophy (DRPLA) is most similar to HD in its clinical features and neuropathology (Ross et al., 1997). Study of the different polyglutamine diseases in parallel is likely to lead to elucidation of shared mechanisms.
Generation of mouse models has played an important role in the study of HD pathogenesis (Menalled and Chesselet, 2002, Rubinsztein, 2002). Transgenic mouse models made using an N-terminal fragment of Htt containing the expanded polyglutamine repeat have yielded a severe behavioral phenotype, with abnormal movements, incoordination, ataxia, and weight loss, progressing to early death (Mangiarini et al., 1996, Schilling et al., 1999a). The intranuclear inclusions were originally discovered in the transgenic mouse models and then later identified in human patient material (Davies et al., 1997, DiFiglia et al., 1997). Transgenic mice, like HD patients, have both intranuclear inclusions and also neuritic and cytoplasmic aggregates. Transgenic and knock-in mouse models have been made expressing full-length Htt with an expanded repeat. To date, these have generally had a less severe behavioral phenotype but have a more restricted neuropathology, including nuclear translocation of mutant Htt (Lin et al., 2001, Menalled et al., 2002, Slow et al., 2003, Wheeler et al., 2000). The transgenic models made with the N-terminal fragment of Htt have generally had more widespread pathology than seen in human cases. A transgenic model with inducible expression of an N-terminal fragment of Htt has also been generated, making it possible to show that there is at least partial reversibility of both the behavioral and neuropathologic phenotype when expression of mutant Htt is switched off (Yamamoto et al., 2000).
Several lines of evidence suggest the possibility that proteolytic cleavage of Htt may contribute to the pathogenesis of HD. The mouse models made with an N-terminal fragment generally have a more severe behavioral and pathologic phenotype than models expressing full-length Htt. In cell model experiments, transfection of N-terminal fragments generally gives greater cell toxicity than transfection of full-length mutant Htt (Cooper et al., 1998, Martindale et al., 1998, Saudou et al., 1998). Most cell model experiments have suggested that translocation of the N-terminal fragment into the nucleus enhances toxicity (Peters et al., 1999, Ross, 2002, Saudou et al., 1998). In addition, N-terminal fragments of Htt have been detected in human postmortem tissue and mouse models, though the length and nature of the fragments have been difficult to characterize precisely (DiFiglia et al., 1997, Gafni and Ellerby, 2002, Goldberg et al., 1996, Kim et al., 2001, Mende-Mueller et al., 2001, Sun et al., 2002, Wellington et al., 2002, Yu et al., 2003). By contrast, other careful examinations of Htt have not found any evidence for cleavage (Wheeler et al., 2000), and it has been suggested that mutant Htt may even be resistant to proteolytic cleavage (Dyer and McMurray, 2001).
Htt can be cleaved in several places by caspase enzymes (Goldberg et al., 1996, Wellington et al., 2002). Caspase cleavage may contribute to toxicity (Ellerby et al., 1999, Wellington et al., 2000). The caspase cleavage sites are located between amino acids 513 and 586. Caspase enzymes that can cleave Htt include caspase-2, -3, -6, and -7 (Hermel et al., 2004, Wellington et al., 1998, Wellington et al., 2000). Htt can also be cleaved in several places by calpains (Kim et al., 2001, Gafni and Ellerby, 2002), which may also contribute to toxicity (Gafni et al., 2004). The major calpain sites are located between amino acids 469 and 536 (Gafni and Ellerby, 2002, Gafni et al., 2004), though other smaller fragments have been described after ischemic injury (Kim et al., 2003). Calpains defined as capable of cleaving Htt include calpains-1 and -2.
In addition, putative N-terminal fragments of Htt smaller than those which would be predicted to arise by caspase or calpain cleavage have been detected in human HD postmortem brain material (DiFiglia et al., 1997) and in at least one knock-in mouse model (Li et al., 2000, Zhou et al., 2003). However, the length and nature of these fragments have been difficult to establish exactly. In a recent cell model study in which a full-length Htt construct with an expanded repeat was inducibly expressed in NG108 cells, at least two smaller cleavage products were detected. The fragments were termed cp-A and cp-B and suggested to arise from sequential cleavage events in the approximate range of amino acids 100–240, with the smaller fragment entering the nucleus, and contributing to the formation of nuclear inclusions and possibly cell toxicity (DiFiglia, 2002, Lunkes et al., 2002).
In the present study, we sought to generate a new mouse model of HD expressing full-length Htt using an inducible promoter so that expression can be controlled in order to facilitate studies of pathogenesis and therapeutics. In addition, we sought to determine whether there might be proteolytic cleavage of Htt in this model and the nature of the fragments that might be generated.
Section snippets
Transgene and other plasmid construction
The pTet-splice vector (Life Technologies, Inc.) was digested with SalI and HindIII, and an SKH linker containing a BspEI site was ligated into the cut pTet-splice vector. The construct was then digested with BspEI. Full-length cDNA of Htt containing 23 consecutive glutamines was excised from a wild-type full-length Htt construct with NotI, and ligated into the BspEI-cut pTet-splice vector (designated as pTet-splice-FL23Q-with-no-tag). To create a myc tag, N-terminal Htt fragments containing
Results
The constructs used for inducible expression in this mouse model are shown in Fig. 1. An N-terminal myc tag was introduced into a full-length Htt construct with 148 consecutive glutamines in order to facilitate identification of N-terminal Htt cleavage fragments. This construct was cloned into the pTet-splice vector, and lines of tet-op huntingtin mice were generated, using standard transgenic technology (Schilling et al., 1999a, Schilling et al., 1999b), incorporating the transgene encoding
Discussion
Our results indicate that we have developed an inducible mouse model of HD expressing full-length Htt with an expanded polyglutamine repeat. The model has a progressive behavioral phenotype, leading to death. Aspects of the phenotype, including abnormal movements, incoordination and ataxia, weight loss, and hindlimb clasping when suspended by the tail, continue to progress to an end stage, in which the animals have a hunched back, are smaller than controls, and are hypo-active. The
Acknowledgments
This work was supported by the National Institutes of Health grants NS16375, NS34172, and NS38144 (CAR) and NS40251A (LME). We are grateful to the Hereditary Disease Foundation and the High Q Foundation for their support to CAR and LME, and to the Huntington's Disease Society of America for their support to CAR (Coalition for the Cure), DRB, and LME. JG is supported by a National Institutes of Health postdoctoral fellowship F32 NS043937.
References (62)
- et al.
A vector for expressing foreign genes in the brains and hearts of transgenic mice
Genet. Anal.
(1996) Transcriptional dysregulation in Huntington's disease
Trends Neurosci.
(2000)- et al.
Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation
Cell
(1997) Huntingtin fragments that aggregate go their separate ways
Mol. Cell
(2002)- et al.
Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity
J. Biol. Chem.
(1999) - et al.
Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus
J. Biol. Chem.
(2004) - et al.
Huntingtin is degraded to small fragments by calpain after ischemic injury
Exp. Neurol.
(2003) - et al.
Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions
Mol. Cell
(2002) - et al.
Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice
Cell
(1996) - et al.
Mouse models of Huntington's disease
Trends Pharmacol. Sci.
(2002)
Nuclear localization of a non-caspase truncation product of atrophin-1, with an expanded polyglutamine repeat, increases cellular toxicity
J. Biol. Chem.
Preparation of human cDNas encoding expanded polyglutamine repeats
Neurosci. Lett.
Nuclear targeting of mutant Huntingtin increases toxicity
Mol. Cell Neurosci.
When more is less: pathogenesis of glutamine repeat neurodegenerative diseases
Neuron
Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington's disease and related disorders
Neuron
Lessons from animal models of Huntington's disease
Trends Genet.
Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions
Cell
Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA
Neuron
Expanded polyglutamine stretches form an ‘aggresome’
Neurosci. Lett.
Transcriptional abnormalities in Huntington disease
Trends Genet.
Decreased cAMP response element-mediated transcription: an early event in exon 1 and full-length cell models of Huntington's disease that contributes to polyglutamine pathogenesis
J. Biol. Chem.
Polyglutamine repeat length-dependent proteolysis of huntingtin
Neurobiol. Dis.
Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract
J. Biol. Chem.
Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells
J. Biol. Chem.
Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease
Cell
The likelihood of being affected with Huntington disease by a particular age, for a specific CAG size
Am. J. Hum. Genet.
Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture
Hum. Mol. Genet.
From neuronal inclusions to neurodegeneration: neuropathological investigation of a transgenic mouse model of Huntington's disease
Philos Trans. R. Soc. Lond., B Biol. Sci.
Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length
J. Neurosci.
Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain
Science
Weight loss in early stage of Huntington's disease
Neurology
Cited by (0)
- 1
Current address: Department of Neuropsychiatry, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama City, Okayama 700-8558, Japan.
- 2
Current address: Department of Neuroscience, Brain Research Institure, Niigata University, I Asahimachi, Niigata 951-8585, Japan.
- 3
Current address: Leibniz Institute for Age Research—Fritz-Lipmann-Institute e.V. (FLI), Beutenbergstr.11, D-07745 Jena.
- 4
Current address: Academic Neurology Unit, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.
- 5
Current address: Santa Fe Health Alzheimer's Research Center, Department of Neuroscience, McKnight Brain Institute, University of Florida, 100 Newell Drive, Rm. L1-100H, PO Box 100244, Gainesville, FL 32610-0244, USA.