Use of Mutant Mice in Studies of Brain Disease

Transgenic mice produced by this method are generally gain-of-function mutants, because the transgene is designed either to express a novel gene product or to disrupt a normal gene product by expressing a "dominantnegative" alternative. It is also possible to put a DNA fragment in an opposite direction and hence to produce transgenic mice expressing antisense RNA, which will reduce gene function (Jouvenceau et al. 1999; Katsuki et al. 1988). Expression of mutant amyloid precursor protein (APP) or presenilin 1 (PS1) in mice has generated many animal models that may be related to Alzheimer's disease. These transgenic mice have facilitated studies of the pathogenesis, molecular mechanisms, and behavioral abnormalities of Alzheimer's disease (Bornemann and Staufenbiel 2000; Janus et al. 2001). Nevertheless, because the transgene integrates into the mouse genome randomly and often exists as several copies, the interpretation of studies with transgenic mice is difficult.

Experiments with knockout mice have provided novel insights into the functional roles of neuronal genes and, in some cases, animal models relevant to brain disorders. The targeted mutants in a gene of interest, however, can sometime lead to embryonic lethality in mice, thus obscuring the particular role of that gene in a target tissue or in the adult. Furthermore, in some instances, genes related to the gene that was eliminated undergo increased expression in the knockout mice. In this case, the related gene compensates for the gene of interest and yields a phenotype that resembles the "normal" animal. This has been seen for the knockouts of the axonal microtubule-associated protein tau (Takei et al. 2000). Here, we take Huntington's disease to exemplify the use of transgenic mice in the study of neurological and psychiatric disease.

Huntington's disease (HD) is a genetic neurological disorder that is inherited in an autosomal dominant manner. The gradual atrophy of the striatum is its pathological hallmark. It has a prevalence of 3-10 affected subjects per 100,000 individuals in Western Europe and North America (Gil and Rego 2008). The first symptoms generally appear in middle age, and the disease is progressive and invariably fatal 15-20 years after its onset (Ho et al. 2001). HD is caused by an expansion of cytosine-adenine-guanine (CAG) repeats in exon 1 of the HD gene. The HD gene is located on the short arm of chromosome 4 (4p63) and encodes the protein huntingtin, composed of more than 3,100 amino acids with a polyglutamine tail, which is widely expressed throughout the body in both neuronal and nonneuronal cells.

The function of huntingtin has been revealed with different research approaches, especially those with transgenic mice. Engineered knockout mice that disrupt exon 4 (Duyao et al. 1995), exon 5 (Nasir et al. 1995), or the promoter (Zeitlin et al. 1995) of mouse gene homology, hdh, showed embryonic lethality. A subsequent study using mutant human huntingtin to compensate for the absence of endogenous huntingtin rescued the embryonic lethality of mice homozygous for a targeted disruption of the hdh gene (Leavitt et al. 2001). These suggest its essential role for normal embryonic development. Moreover, condition knockout mice indicated that huntingtin is also required throughout life, because adult mice are sterile, develop a progressive motor phenotype, and with short life span after inactivating hdh gene during adulthood (Dragatsis et al. 2000). Furthermore, overexpression of wild-type huntingtin, bearing 12 glutamine residues, in transgenic mice brought significant protection against apoptosis triggered by NMDA (Leavitt et al. 2006), suggesting that huntingtin may play a role in cellular apoptosis.

As mentioned above, HD is a neurodegenerative disorder caused by uninterrupted CGA trinucleotide repeats that located near the 5'-end in exon 1. Consequently, mutant huntingtin bears a string of consecutive glutamine residues in the NH2-terminal, 17 amino acids downstream of the initiator (Gil and Rego 2008). The length of glutamine residues in NH2-terminal of mutant huntingtin is the primary and predominant determinant for severity of HD. To elucidate the mechanism of neurodegeneration in HD, multiple mouse models of HD have been established. These models vary in terms of the site of transgene incorporation, promoter used, gene expression levels, CAG repeat length, and background mouse strain used. These transgenic models have facilitated investigations into potential pathogenic mechanisms of HD. At present, there are three categories of mouse model:

1. Mice expressing exon-1 fragments of human huntingtin gene (HD) containing polyglutamine mutations (R6/1, R6/2, and R6/5). This transgenic mouse carries exon 1 of the HD gene with 115-155 CAG repeats. The transgene protein contains the first 69 amino acids of huntingtin in addition to the number of residues encoded by the CAG repeats. Extensive neuropathological analysis has been performed on the brains is R6/2 mice. The mice display subtle motor and learning deficits at approximately 1 month and overt symptoms appear by 2 months, and they usually die at 3 or 4 months (Carter et al. 1999; Lione et al. 1999; Murphy et al. 2000). It is worthy to note that the characteristic nuclear inclusions were first detected with antibodies against the W-terminal portion of huntingtin in R6/2 mice. R6/2 mice show measurable deficits in motor behavior that increase progressively until death. R6/2 mice are a model of HD to aim at studying the severity of motor symptoms or the course of the disease. In R6/2 mice, many neurotransmitter receptors, such as NMDA, AMPA, mGluR2, DA, and GABA, display abnormal response to their ligands (Ali and Levine 2006; Cepeda et al. 2004; Cha et al. 1998; Dunah et al. 2002; Starling et al. 2005).

2. Mice expressing the full-length human HD gene. Yeast artificial chromosomes (YACs) were used to create a YAC transgenic mouse model of HD that expresses the full-length human HD gene. The transgenic mice express human transgenic huntingtin with 18, 46, 72, or 128 polyglutamine repeats. The YAC mice with 72 CGA repeats develop a progressive motor phenotype, neuronal dysfunction, and selective striatal neurodegeneration similar to that seen in HD by a 12-month timeline. YAC mice with 128 CAG repeats (YAC128 mice) exhibit initial hyperactivity, followed by the onset of motor deficits and finally hypokinesis, which show phenotypic uniformity with low interanimal variability present. The motor deficit in the YAC128 mice is highly correlated with striatal neuronal loss, providing a structural correlate for the behavioral changes (Hodgson et al. 1999; Slow et al. 2003). These lines of transgenic mice may be extremely useful for preclinical experimental therapeutics.

3. Knock-in HD transgenic mice. In knock-in mice, a mutated DNA sequence is exchanged for the endogenous sequence without any other disruption of the gene. To establish the line of knock-in HD mice, CGA repeats in the murine hdh gene were replaced with human mutant CAG repeats. These mice are characterized by a biphasic progression in behavioral anomalies, and the nuclear inclusions appear late and are preceded by nuclear staining for huntingtin, followed by the presence of microaggregates of the mutant protein in the nucleus and the neuropil (Menalled et al. 2002, 2003, 2005). These knock-in mice are considered to be an ideal genetic model of HD to evaluate the effectiveness of new therapies and to study the mechanisms involved in the neuropathology of HD.

The above examples illustrate both the potential and the current promise of manipulation of the mouse genome for the study of human neuropsychiatric disease.

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