Screening of Therapeutic Strategies for Huntington's Disease in YAC128 Transgenic Mice


Joana M. Gil-Mohapel, Ph.D., Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, BC, V8W 2Y2, Canada.
Tel.: +1.250.472-5547;
Fax: +1.250.472-5505;


Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by an unstable expansion of cytosine-adenine-guanine (CAG) repeats in the HD gene. The symptoms include cognitive dysfunction and severe motor impairment with loss of voluntary movement coordination that is later replaced by bradykinesia and rigidity. The neuropathology is characterized by neuronal loss mainly in the striatum and cortex, and the appearance of neuronal intranuclear inclusions of mutant huntingtin. The mechanisms responsible for neurodegeneration are still not fully understood although excitotoxicity and a consequent increase in intracellular calcium concentration as well as the activation of caspases and calapins are known to play a key role. There is currently no satisfactory treatment or cure for this disease. The YAC128 transgenic mice express the full-length human HD gene with 128 CAG repeats and constitute a unique model for the study of HD as they replicate the slow and biphasic progression of behavioral deficits characteristic of the human condition and show striatal neuronal loss. As such, these transgenic mice have been an invaluable model not only for the elucidation of the neurodegenerative pathways in HD, but also for the screening and development of new therapeutic approaches. Here, I will review the unique characteristics of this transgenic HD model and will provide a summary of the therapies that have been tested in these mice, namely: potentiation of the protective roles of wild-type huntingtin and mutant huntingtin aggregation, transglutaminase inhibition, inhibition of glutamate- and dopamine-induced toxicity, apoptosis inhibition, use of essential fatty acids, and the novel approach of intrabody gene therapy. The insights obtained from these and future studies will help identify potential candidates for clinical trials and will ultimately contribute to the discovery of a successful treatment for this devastating neurodegenerative disorder.


Our understanding of Huntington's disease (HD) pathogenesis as well as the ability to develop novel therapies, depends on the availability of suitable models of the disease. Therefore, modeling HD in transgenic rodent models is useful for understanding the relation between neuronal dysfunction and/or death and the development of abnormal behaviors. Moreover, transgenic rodent models also provide excellent tools to test the efficiency of new therapeutic approaches [for review, see 1,2].

There are several rodent models available for the study of HD. These models primarily differ in the size of the expressed huntingtin fragment, the number of cytosine-adenine-guanine (CAG) repeats, the promoter driving the transgene (and consequently the expression of the mutant protein), and the background strain. As a consequence, each model exhibits unique phenotypes [for review, see 3].

Undoubtedly, the HD transgenic models that have been most extensively studied are the R6 mouse lines produced in 1996 by Mangiarini et al. [4]. However, their accelerated phenotype [4] and limited striatal cell loss [5–7] may not reflect the actual human condition and, in some circumstances, reduce the ability to detect subtle improvements due to a specific therapy. Moreover, the number of CAG repeats that are expressed in these mice (approximately 115 and 150 repeats in the R6/1 and R6/2 lines, respectively) do not mimic the number of repeats (approximately 39) that cause the most common adult-onset form of the disease in humans [for review, see 2]. In fact, in humans longer repeats are normally associated with high allele instability during paternal transmission and result in juvenile and infantile cases that are characterized by a more severe phenotype and a faster progression of the disease [for review, see 8]. It is also important to note that therapies that are predicted to function at the level of the full-length mutant huntingtin are unlikely to be optimal to test in this model, which expresses a truncated form of the mutant protein [for review, see 2].

More recently though, various transgenic mouse models have been generated using the full-length human HD gene with different CAG lengths as the transgene. These include the bacterial artificial chromosome (BAC) and the yeast artificial chromosome (YAC) HD mouse models. BAC HD transgenic mice express full-length mutant huntingtin with 97 CAG repeats under the control of the endogenous huntingtin regulatory machinery [9]. These mice exhibit progressive motor deficits, neuronal synaptic dysfunction, and late-onset selective neuropathology, which includes significant cortical and striatal atrophy and striatal dark neuronal degeneration [9,10]. In these mice, the slow neuronal degeneration is elicited by full-length mutant huntingtin and a small amount of toxic N-terminal fragments, without early nuclear accumulation of aggregated mutant huntingtin [9].On the other hand, YAC mice expressing the full-length HD gene with 46, 72 [11], or 128 [12] CAG repeats under the control of the endogenous huntingtin promoter and its regulatory elements display a selective degeneration of striatal medium-sized spiny neurons (i.e., the neuronal population most affected in HD) and develop their phenotype over the course of 12–18 months [12]. Importantly, this specific HD-like phenotype is not caused by the YAC itself, as YAC mice expressing the human HD gene with 18 CAG repeats (i.e., wild-type human huntingtin) do not develop the disease and are indistinguishable from normal wild-type mice [11]. Interestingly, as is the case for the R6 mice, the numbers of CAG repeats expressed both in the BAC and the YAC HD mouse models also do not reflect the most common human mutation. This is due to the fact that to observe HD-like symptoms in mice, the CAG repeat stretch has to be longer than the ones that cause adult-onset HD in humans. However, the disease progression in the full-length HD mouse models (BAC and YAC HD mice) is quite different from the fast and severe progression observed in the R6 lines, suggesting that factors other than the length of the CAG repeat stretch are responsible for the rate of disease progression. One possible cause for this difference may be the size of the transgene expressed by the different models. Indeed, the R6 lines only express a small truncated fragment of the human huntingtin gene [4] as opposed to the BAC [9] and YAC [12] mice, which express the full-length gene. Thus, it is possible that in the R6 mice the disease is manifested earlier as these animals express the fragments that are thought to be responsible for the disease while in the full-length HD mouse models the additional steps required to produce the toxic fragments from the full-length protein (i.e., cleavage of huntingtin by caspases and calpains; 13–16) may delay the onset of the symptoms and the progression of the disease. In particular, the YAC128 HD mice have been extensively studied and characterized and because these mice present several advantages when compared with the R6 lines, many groups have now adopted this model as their preferred choice for the screening of new therapies for HD.

The YAC128 HD Transgenic Mice

In an effort to create a YAC mouse with both an accelerated and quantifiable phenotype, Hodgson et al. extended their previous research [11] and generated a YAC transgenic mouse model expressing the full-length human HD gene with 128 CAG repeats (YAC128) [12].

These mice develop behavioral abnormalities that follow a byphasic pattern with an initial phase of hyperactivity followed by the onset of motor deficits, which can be detected as early as 2 months and clearly by 4 months of age and finally by hypokinesis [17–21]. Importantly, there is a positive correlation between the onset of motor deficits and the extent of striatal neuronal loss in these mice, which provides a neuropathological cause for the observed behavioral deficits [12]. Furthermore, YAC128 transgenic HD mice also develop mild cognitive deficits, which precede the onset of motor abnormalities and can be detected as early as 2 months of age and progressively deteriorate with age [20] as well as a depressive-like behavior at the early stage of 3 months of age [22].

At the neuropathological level, it is possible to detect a significant atrophy of the striatum, globus pallidus and cortex with relative sparing of the hippocampus and cerebellum in the brains of YAC128 mice as early as 9 months of age [12,23]. In agreement with the gross atrophy of these brain structures, neuronal loss can be detected in the striatum and cortex, the two brain regions most affected in HD patients, but not in the hippocampus of 12 months old YAC128 mice [23]. Importantly, YAC128 mice also display enhanced sensitivity to multiple excitotoxins in the early phase of the disease, prior to development of motor abnormalities [24,25]. However, by 10 months of age these mice become resistant to excitotoxic stress [25]. This byphasic response to excitotoxins as well as an abnormal and selective increase in striatal glutamate receptor subunit expression and binding [26], strongly suggest that striatal cell death in HD is mediated, at least in part, through an excitotoxic mechanism that might be caused by a dysfunction of the glutamatergic corticostriatal pathway [27,28, for review, see 8].

Aggregation of mutant huntingtin and the consequent formation of neuronal intranuclear inclusions has been considered as an hallmark of HD [for review, see 8] and a widespread distribution of these inclusions has been consistently observed in the striatum and cortex of both human HD patients [29] and R6 mice [30]. In the YAC128 HD mouse model, it is possible to observe an increase in nuclear huntingtin immunoreactivity at 12 months of age [12,23] and clear inclusions can be detected in striatal cells in older end-stage animals (i.e., at 18 months of age) [12]. Importantly, the fact that YAC128 HD mice present significant neuronal dysfunction and loss prior to the appearance of clear intranuclear inclusions of mutant huntingtin strongly supports the idea that inclusions may represent a side effect of the ongoing cellular dysfunction, or may even exert a protective role during the early stages of the disease [31–33, for review, see 8]. In further agreement with this hypothesis, ”shortstop” YAC transgenic mice (which express a truncated fragment of the human HD gene with approximately 120 CAG repeats) show widespread neuronal intranuclear inclusions at a very early age. However, these mice display no features of neuronal dysfunction and/or degeneration further indicating that inclusions of mutant huntingtin may not be pathogenic in vivo[34].

At the peripheral level, YAC128 mice show an increase in body weight [34], which appears to be correlated with the plasma levels of insulin growth factor-1 (IGF-1), as treatment with 17-beta-estradiol reduces plasma IGF-1 levels and decreases their body weight to wild-type levels [35]. YAC128 mice also display testicular atrophy and degeneration [21,36] with no apparent decrease in testosterone levels or loss of gonadotropin-releasing hormone-containing neurons in the hypothalamus [21]. Interestingly, this symptom seems to be aggravated in animals that do not express wild-type huntingtin [36]. Furthermore, a deficit in survival has been detected in male YAC128 mice, a symptom that is exacerbated by the loss of wild-type huntingtin [36] indicating that the HD phenotype also depends on the expression levels of the mutant protein. To date, no studies have investigated whether female YAC128 mice also show deficits in survival. Therefore, a further characterization of the HD female phenotype is warranted.

Therapeutic Strategies

Although clinically relevant, it is particularly challenging to perform therapeutic trials in HD patients [for review, see 2]. Thus, it is extremely important that all novel therapies for HD are extensively studied in in vivo models of the disease before being tested in patients. As stated above (section 1), while R6 mice only express a truncated fragment of the mutated human huntingtin gene and display an accelerated phenotype with limited striatal neuronal loss [4; for review, see 2], the YAC128 mice express full-length human mutant huntingtin and show a slower disease progression with specific striatal neurodegeneration [12]. Furthermore, the YAC128 HD phenotype described above shows phenotypic uniformity with low interanimal variability, which makes this transgenic mouse model a more accurate representation of the human condition and therefore a better candidate for therapeutic trials (see Table 1 where the various therapeutic strategies that have been tested in the YAC128 mice are listed and their efficacy in this model is compared with the results obtained in other HD mouse models).

Table 1.  Therapeutic strategies for HD that have been tested in the YAC128 full-length transgenic mouse model
Treatment (Reference)DoseDurationEffect on behaviorEffect on neuropathologyEffect on other HD transgenic models (Reference)Human clinical trials [Reference]
Overexpression of wild-type huntingtin [37]From birthNo effect↓ Striatal neuronal atrophy
Memantine [40,41] blockage of extrasynaptic NMDA receptors1 mg/kg drinking water2–12 months 2–4 months↑ Rotarod performance ↓ Motor learning deficits↑ Inclusions ↑ Striatal volume ↓ CREB signaling deficits↓ Motor decline ↓ Chorea [42,43]
Cystamine [19] transglutaminase inhibition225 mg/kg drinking water7–12 monthsNo effect↑ Striatal volume ↓ Striatal neuronal loss and atrophyR6/2 ↑ Life span Behavioral and neuropathologic improvement [66–68]Well tolerated at 20 mg/Kg/day [73]
GFP-IC10 [46] adeno-associated virus expressing a cytosolic C-terminal tail of the InsP3R2.0 × 1011 TU/mLInjection at 2 months↑ Balance beam coordination ↑ Footprint pattern↓ Striatal neuronal loss and atrophy ↓ Nuclear huntingtin
Tetrabenazine [87] dopamine inhibitor0.3 mg gavage (2 × week)2–10 months↑ Rotarod performance ↑ Balance beam coordination ↑ Footprint pattern↓ Striatal neuronal loss↓ Chorea [88,89]
Expression of caspase-6-resistant mutant huntingtin [17,22]From birthNo motor deficits no depressive phenotypeNo striatal degeneration resistance to excitotoxicity
Ethyl-EPA [100] essential fatty acid1% in chow7–12 months↑ Rotarod and open-field performanceNo effectMotor improvement (UHDRS) [101]
AAV-Happ1 [116] adeno-associated virus expressing an intrabody against the huntingtin N-terminus1.0 × 1013 TU/mL (1 μL)Injection at 2 months↑ Rotarod and open-field performance ↑ Balance beam coordination and climbing performance ↑ Performance in cognitive tests↓ Enlargement of lateral ventriclesLentiviral model R6/2 mice N171–82Q mice BAC mice behavioral and neuropathologic improvement [116]

Protective Role of Wild-Type Huntingtin

It has been proposed that the loss of wild-type huntingtin (which is now known to be implicated in the regulation of several prosurvival intracellular signaling cascades and in the modulation of antiapoptotic pathways) can contribute to the HD phenotype [for review, see 8]. In order to test this hypothesis in vivo, Van Raamsdonk et al. generated YAC128 mice that do not express wild-type human huntingtin (YAC128−/−) but express the same amount of mutant human huntingtin as normal YAC128 HD mice (YAC128+/+, which express both the wild-type and the mutant alleles) and compared their phenotypes [36]. Interestingly, YAC128−/− HD mice perform worse in the rotarod test and are more hypoactive than YAC128+/+ HD mice. Moreover, absence of wild-type human huntingtin increases testicular atrophy and has a negative effect on the life span of male YAC128 HD mice. However, striatal neuropathology (namely striatal volume, number of striatal neurons, and expression of dopamine and cyclic adenosine monophosphate-regulated phosphoprotein of 32 KDa, DARPP-32) was not affected by the lack of wild-type huntingtin, although a modest decrease in striatal neuronal atrophy was observed [36]. Also, crossing YAC128 HD mice with mice that overexpress wild-type human huntingtin (YAC18 mice) in order to generate YAC128 HD mice that overexpress the wild-type protein (YAC18/128 mice) had no effect on the development of the characteristic YAC128 HD behavioral phenotype (as assessed by the performance on the rotarod and the automated open field tests). Moreover, YAC18/128 double-transgenic HD mice show no significant improvement in striatal volume, striatal neuronal numbers, or striatal DARPP-32 expression when compared to YAC128 HD mice. Again, overexpression of wild-type huntigtin only had a beneficial effect at the level of striatal neuronal atrophy [37]. Given these results, it is reasonable to speculate that the striatal HD neuropathology is caused primarily by the toxicity of the mutant protein itself and that inducing the expression of wild-type huntingtin might not be sufficient to treat HD.

Protective Role of Mutant Huntingtin Aggregation and Prevention of Excitotoxicity

Excitotoxicity has been implicated in HD [for review, see 8], and several in vitro and in vivo studies have strongly suggested that an increase in striatal excitotoxicity is responsible, at least in part, for the loss of neurons observed in this brain region in the YAC128 mouse [24–26,38,39]. Interestingly, besides their role in mediating excitotoxic cell death, it is now known that N-methyl-D-aspartate (NMDA) glutamate receptors can also regulate the mechanisms involved in the formation of intranuclear inclusions of mutant huntingtin depending on their localization (synaptic vs. extra-synaptic). Indeed, synaptic NMDA receptor activity can promote the formation of inclusions via a T complex-1 ring complex-dependent mechanism, thus rendering neurons more resistant to cell death [40], which is in agreement with the proposed neuroprotective role of inclusions in HD [24,31–33; for review, see 8]. On the other hand, stimulation of extra-synaptic NMDA receptors increases vulnerability to neuronal death by impairing the neuroprotective cyclic adenosine monophsophate response element-binding protein (CREB)-peroxisome proliferator-activated receptor-gamma coactivator-1-alpha cascade and increasing the level of the small guanine nucleotide-binding protein Rhes, which is known to SUMOylate and disaggregate mutant huntingtin, and thus decrease the inclusion load [40]. In agreement, a specific increase in extra-synaptic NMDA receptor expression and function and a consequent decrease in nuclear CREB activation has been detected in the striatum of YAC128 mice. These changes are observed in the absence of dendritic morphological alterations and can be detected before the onset of the disease. Moreover, the dysregulation of this pathway is aggravated by an increase in the number of CAG repeats expressed (as assessed by comparing striatal neurons from YAC76 and YAC128 mice), and is dependent on caspase-6-mediated cleavage of mutant huntingtin, because caspase-6-resistant YAC128 striatal neurons do not show such alterations [41]. According to these findings, long-term blockage of extra-synaptic NMDA receptor activity might have therapeutic benefits, whereas simultaneous blockage of synaptic and extra-synaptic NMDA receptor activity might prove to have long-term deleterious effects, as blockage of synaptic activity would eventually lead to neuronal death through an increase in intracellular soluble mutant huntingtin. Indeed, treatment of YAC128 mice from 2 to 4 months of age with a low dose (1 mg/kg) of memantine to block extra-synaptic (but not synaptic) NMDA receptor activity reversed the CREB signaling deficits as well as the early motor-learning deficits (i.e., induced a decrease in the number of falls when learning the rotarod test) [41]. However, the authors did not analyze whether increasing inclusion formation through treatment with memantine could also delay the onset of the hyperkynesic phenotype in young YAC128 mice. Nevertheless, when treatment was continued until 12 months of age, YAC128 mice showed increased inclusion formation, reduced loss in striatal volume, and improved motor performance (as assessed by the rotarod test) [40]. On the other hand, a high dose of memantine (30 mg/kg), which blocks both extra-synaptic and synaptic NMDA receptor activity, decreased the number of neuronal inclusions and worsened both the neuropathological (i.e., striatal volume) and behavioral (i.e., locomotor activity) outcomes [40]. Taken together, these results further support the involvement of excitotoxicity in the neuropathology of HD, as extra-synaptic NMDA receptor activation results in the accumulation of N-terminal toxic fragments of mutant huntingtin. Furthermore, these findings support the neuroprotective role of intranuclear inclusions of mutant huntingtin and strongly suggest that maintenance of synaptic activity with abrogation of extra-synaptic activity can be of potential therapeutic relevance for HD. Indeed, two clinical trials that analyzed the potential therapeutic benefits of memantine treatment in HD patients have already been performed. In the first one, the effectiveness of this compound (up to 30 mg/day) with regard to retardation of the disease progression was examined in 27 patients in a 2-year multicenter trial. No motor decline was observed at the 12 and 24 months evaluations, strongly suggesting that memantine may be useful in the retardation of the progression of this neurodegenerative disorder [42]. In agreement, in a subsequent pilot trial that included 12 patients, memantine (20 mg/day) significantly improved the motor symptoms (including chorea) although no effects on the cognitive, behavioral, functional, and independence ratings were detected [43].

Inhibition of Intracellular Calcium Rise

Tang et al. have sought to investigate the relationship between glutamate-induced calcium signaling and apoptosis in cultured YAC128 striatal medium-sized spiny neurons. Importantly, this study revealed that glutamate-induced apoptosis was mediated by the activation of metabotropic glutamate receptors 1/5 and the NMDA receptor NR2B, an increase in intracellular calcium concentration with a consequent calcium overload of the mitochondria and the opening of the mitochondrial permeability transition pore, which in turn triggers the mitochondrial apoptotic pathway (i.e., release of cytochrome c and activation of caspases). Furthermore, the mitochondrial calcium uniporter blocker Ruthenium 360 as well as the mitochondrial permeability transition pore blockers bongkrekic acid, Nortriptyline, Desipramine, Trifluoperazine, and Maprotiline were all neuroprotective and were able to prevent cell death. Interestingly, a protective effect was also observed in the presence of 2-APB and Enoxaparin (Lovenox), two membrane-permeable blockers of the inositol 1,4,5-trisphosphate receptor (InsP3R) [44], which is in agreement with a previous study indicating that mutant huntingtin specifically binds to and activates this intracellular calcium release channel [45]. Together, these results strongly suggest that both calcium and mitochondrial permeability transition blockers may have therapeutic potential in the treatment of HD. However, the actual therapeutic value of these drugs can only be determined upon the completion of properly controled in vivo studies. In agreement, in order to further clarify the potential therapeutic value of InsP3R blockers, the same authors have recently generated lentiviral and adeno-associated viruses expressing a cytosolic C-terminal tail of the InsP3R containing 122 amino acids fused with green fluorescence protein (GFP-IC10) and tested their therapeutic efficacy using the YAC128 HD transgenic mouse model [46]. In agreement with the initial study, infection of cultured YAC128 medium-sized spiny neurons with Lenti-GFP-IC10 virus stabilized calcium signaling and protected against glutamate-induced apoptosis. Furthermore, intrastriatal injections of Adeno-GFP-IC10 virus significantly alleviated motor deficits (as assessed by the balance beam coordination assay and analysis of the footprint pattern) and reduced the loss and shrinkage of striatal neurons as well as nuclear huntingtin immunoreactivity in 12 months old YAC128 mice [46]. Together, these results further validate InsP3R as a potential therapeutic target for HD.

Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), has been used as a herbal medicine in Asia in the treatment of several neurodegenerative and aging-related disorders. Ginsenosides are the main components responsible for the actions of ginseng and more than 30 different ginsenosides have been already isolated and identified. Previous studies have demonstrated that certain ginsenosides can protect cortical [47] and hippocampal [48] neurons from glutamate- [47,48] and NMDA-induced [48] cell death by preventing an increase in intracellular calcium concentration. Moreover, the ginsenosides Rb1, Rb3, and Rd have been shown to be neuroprotective in the 3-nitropropionic acid rat model of striatal neurodegeneration [49,50]. Recently, Wu et al. have also reported that the ginsenosides Rb1, Rc, and Rg5 effectively protect cultured YAC128 striatal medium-sized spiny neurons from glutamate-induced apoptosis by inhibiting the typical glutamate-induced increase in intracellular calcium concentration [51]. Whether this protective effect indicates that these compounds will also mitigate the behavioral and neuropathological symptoms of these mice is an interesting hypothesis that deserves further investigation.

Transglutaminase Inhibition

Transglutaminase catalyses the formation of gamma–glutamyl isopeptide bonds between polyglutamine tracts and lysine residues and thus it has been suggested that this enzyme might be involved in the formation of intranuclear inclusions of mutant huntingtin in the HD brain [52]. In agreement, an increase in transglutaminase activity has been observed in the brains of HD patients [53,54]. However, the number of striatal and cortical aggregates were found to be increased in both R6/1 [55] and R6/2 [56] HD mice upon ablation of “tissue” transglutaminase expression. Nevertheless, despite an increase in the number of huntingtin aggregates, the absence of transglutaminase was associated with an improvement of the motor symptoms and an increase in the life span of both R6/1 [55] and R6/2 [57] mice. These studies further support the idea that aggregate formation may be a protective mechanism of the cell against the more toxic effects of the soluble oligomeric intermediates of the mutant protein. Furthermore, these studies also favor the hypothesis that the primary role of this enzyme in HD pathology may not be mediated by the formation of nuclear inclusions. Alternatively, transglutaminase may contribute to HD pathology through its role in apoptosis. Indeed, overexpression of ”tissue” transglutaminase results in an increased sensitivity to apoptosis [58,59], while decreasing the expression of this enzyme prevents cell death [60]. In addition, transglutaminase may also exacerbate mutant huntingtin toxicity by further promoting the modification of proteins that interact with it [61].

Cystamine is a classical transglutaminase inhibitor that has been shown to decrease polyglutamine aggregation in vitro[62,63] as well as transglutaminase activity and apoptosis induced by either glutamate [64] or an N-terminal fragment of mutant huntingtin [65]. Furthermore, treatment of R6/2 HD mice with this compound has been shown to increase their life span and improve some of their behavioral and neuropathological features [66–68], through a mechanism that does not seem to be mediated by inhibition of transglutaminase activity, as similar beneficial effects have also been found in cystamine-treated R6/2 mice that do not express ”tissue” transglutaminase [57]. Indeed, it might be that the protective effects of cystamine in vivo are not directly linked with transglutaminase inhibition, but rather with its reported antioxidant [69] and antiapoptotic [70] properties, as well as its ability to increase the expression of heat-shock proteins [67] and/or brain-derived neurotrophic factor levels [71]. However, it has been shown that the brain levels of cystamine in cystamine-treated YAC128 HD mice are too low to significantly affect intracellular enzymes that require a critical cysteine residue for activity, thus ruling out the possibility that cystamine treatment is beneficial in HD mice by inhibiting caspases [72].

Given the potential of cystamine as a therapeutic strategy for HD, Pinto et al. have also investigated the effects of cystamine treatment on development of the YAC128 phenotype and striatal neuronal loss. Interestingly, they found an increase in transglutaminase activity in the forebrain of YAC128 HD mice when compared with their wild-type littermate controls that was reduced upon cystamine treatment (starting at 7 months of age). Furthermore, cystamine prevented striatal neuronal loss and decreased the reduction in striatal volume as well as striatal neuronal atrophy. However, this therapeutic strategy had no effect on the downregulation of DARPP-32 expression in striatal neurons and it was unable to prevent the motor dysfunction (i.e., decreased performance on the rotarod and activity on the open field) characteristic of these HD mice [19]. While the reasons for the discrepancies that were found between the R6 and the YAC128 HD models regarding the effects of cystamine treatment on behavioral outcomes are not clear, the beneficial effects that were observed at the neuropathological level in both models strongly suggest that cystamine might be a good candidate for clinical trials in HD patients. Indeed, an initial trial has already been conducted in order to clarify tolerability and dosing of this drug in HD patients. The study was performed in nine HD-affected individuals and demonstrated that cystamine can be tolerable at a dose of 20 mg/kg/day and that nausea and motor impairment were the dose-limiting side effects [73]. A follow-up detailed report on the long-term effects of this treatment is now warranted.

Inhibition of Dopamine Toxicity

In addition to glutamatergic stimulation from the cortex, the striatum also receives dopaminergic input from the substantia nigra pars compacta. Several studies have shown degeneration of these nigrostriatal projections [74,75], an atrophy of dopaminergic neurons in the substantia nigra [76,77], and a striking reduction of the dopaminergic striatal neuronal population in HD brains [78]. Aggregates of mutant huntingtin were also found in this brain region [79]. Furthermore, a marked loss of tyrosine hydroxylase (the rate-limiting enzyme for dopamine biosynthesis) [77] and a downregulation of the dopamine transporter and the D1 and D2 dopamine receptors occurs in HD brains [74,80] and R6/2 mice [81], probably due to altered gene transcription induced by mutant huntingtin. Interestingly, loss of D2 dopamine receptor was suggested to be a sensitive indicator of early neuronal impairment in preclinical carriers of the HD mutation [82]. Furthermore, in vitro and in vivo studies have also shown that dopamine itself can enhance polyglutamine toxicity [83,84], be a source of reactive oxygen species, and a trigger of oxidative stress [for review, see 85].

In agreement with this idea, Tang et al. have recently demonstrated that persistent elevation of striatal dopamine levels exacerbated the behavioral motor deficits and the neurodegeneration of striatal medium-sized spiny neurons in YAC128 HD mice. Tetrabenazine is a synthetic benzoquinolizine derivative, which binds reversibly to vesicular monamine transporters (VMATs), especially to VMAT2, which is expressed predominantly in the brain. Binding of tetrabenazine to VMAT2 results in the inhibition of monoamines uptake into synaptic vesicles and in a consequent reduction of their release. Furthermore, tetrabenazine also blocks postsynaptic dopamine receptors, thus acting both pre- and postsynaptically [86]. Interestingly, treatment with this clinically relevant dopamine inhibitor (from 2 to 10 months of age) alleviated the motor deficits (as assessed by the rotarod, the beam walking, and the footprint tests) and reduced striatal cell loss in this mouse model of HD [87]. These results further suggest that the nigrostriatal dopaminergic signaling pathway may act synergistically with the glutamatergic corticostriatal pathway to promote striatal cell loss in HD and that antagonists of the dopamine pathway such as tetrabenazine may have therapeutic potential for the treatment of this neurodegenerative disorder. Indeed, a recent 12-week clinical trial demonstrated that tetrabenazine can efficiently reduce chorea in HD ambulatory patients [88,89]. Further studies are needed in order to determine whether a long-lasting improvement can be obtained with a longer treatment regime.

Inhibition of Caspases and Calpains

Mutant huntigtin is cleaved by caspases-3 and -6 both in vitro[13,90,91] and in vivo[14;17;92] and an increase in the active forms of these proteases has been detected in human HD brains [93]. In addition, mutant huntingtin toxicity can be blocked in vitro upon site-directed mutagenesis of all its caspase cleavage sites [94]. These observations raised the possibility that mutant huntingtin fragments that result from caspase-mediated cleavege may be important in the pathogenesis of HD and that inhibiting cleavage of mutant huntingtin by caspases may have beneficial effects. In support of this hypothesis, YAC128 mice expressing a full-length mutant huntingtin construct that is resistant to cleavage by caspase-6 (but not by caspase-3) do not develop motor deficits and striatal neurodegeneration, are resistant to excitotoxicity induced by quinolinic acid [17] and to extra-synaptic NMDA receptor activity (see section 3.2; 41). These results strongly suggest that caspase-6-mediated proteolysis of full-length mutant huntingtin selectively mediates susceptibility to excitotoxic stress in the YAC128 mouse model. Furthermore, the YAC128 depressive phenotype (see section 2.2.1) was also completely rescued in these caspase-6-resistant YAC128 mice [22]. Thus, therapies aimed toward inhibition of huntingtin cleavage by caspase-6 (such as using specific inhibitors of this caspase) are likely to have therapeutical benefits in patients with HD.

Calpains, calcium-activated cysteine proteases that are involved in neuronal apoptosis, have also been detected in the striatum of HD knock-in mice and postmortem HD human brains [15,16]. Furthermore, huntingtin is also a substrate for calpains and mutation of the two calpain-cleavage sites abolishes huntingtin aggregation and toxicity in vitro[16]. In agreement with these initial observations, Cowan et al. have recently reported an increase in calpain activity in cultured YAC128 medium-sized spiny neurons. Importantly, these authors also found that treatment with a calpain inhibitor reduced NMDA-induced apoptosis in both YAC72 and YAC128 striatal medium-sized spiny neurons [39]. These results strongly suggest that the sensitivity of striatal medium-sized spiny neurons to excitotoxicity is mediated, at least in part, by calpain-induced cell death and that calpain inhibitors might have therapeutic potential for the treatment of HD.

Essential Fatty Acids

In line with mitochondrial dysfunction and oxidative stress playing a role in HD [for review, see 8], increasing the antioxidant defenses may have therapeutic value, and several antioxidant compounds have been proven to be beneficial in mitigating some of the neuropathological and behavioral symptoms of the R6 mice [for review, see 2]. In particular, treatment of the R6/1 HD mouse model with a mixture of essential fatty acids was shown to improve motor function and increase the survival of these mice [95].

Eicosapentaenoic acid (EPA) is a naturally obtained omega-3 fatty acid that can also be produced by hydrolysis of the prodrug ethyl-EPA, a compound that has been previously shown to inhibit apoptosis [96], reduce inflammation [97,98], and to improve mitochondrial function [99]. Importantly, administration of ethyl-EPA to symptomatic YAC128 mice beginning at 7 months of age increased their membrane EPA levels and resulted in a significant improvement of their motor dysfunction (as assessed with the open-field and rotarod tests) by 12 months of age. However, the development of the typical striatal neuropathology (i.e., a decrease in striatal volume, striatal neuron counts, striatal neuronal cross-sectional area, and striatal DARPP-32 expression) was not affected by ethyl-EPA treatment [100]. Interestingly, in agreement with this study, symptomatic HD patients that were treated with ethyl-EPA only showed mild improvements on the motor component of the Unified HD Rating Scale (UHDRS) [101]. Thus, although the use of essential fatty acids may not be the best therapeutic approach for the treatment of HD, the fact that the YAC128 mouse model replicated the modest motor improvement observed in ethyl-EPA-treated HD patients strongly suggests that screening potential therapeutic targets in these mice may indicate good candidates for the development of future human clinical trials.

Intrabody Gene Therapy

Intracellular single-chain Fv and single-domain antibodies, or intrabodies, have the potential to alter the folding, interactions, modifications, or subcellular localization of their targets when expressed intracellularly in mammalian cells. Although the formation of mutant huntingtin aggregates is believed to be neuroprotective (by reducing the amount of free toxic N-terminal fragments of the mutant protein) (see section 3.2), it is likely that with the progression of the disease, an increase in aggregation might contribute to neuronal dysfunction in the affected regions of the HD brain (for review, see 8). Indeed, several transcription factors including CREB-binding protein [102,103], specific protein-1 [104,105], and the TATA-binding protein associated factor [105] have been shown to be recruited into neuronal intranuclear inclusions, thus contributing to transcriptional dysregulation in HD. Furthermore, inclusions may physically block the proteasome, preventing the entrance of further substrates into this complex and overloading this important intracellular system of protein degradation (for review, see 106). Finally, an increase in aggregation can also cause the dysregulation of axonal transport by physically blocking narrow axonal terminals (for review, see 107). Thus, intrabody therapy might be of potential therapeutic value for the treatment of this disorder [for review, see 108,109]. In agreement with this idea, previous studies have shown that treatment with anti-N-terminal huntingtin intrabodies reduces aggregation [110–112] and cell toxicity in cell models of HD [111,113] and suppresses neuropathology in an HD Drosophila model [114,115]. Recently, Southwell et al. have also tested the effects of two different intrabodies, V(L)12.3 (an intrabody recognizing the N-terminus of huntingtin) and Happ1 (an intrabody recognizing the huntigtin proline-rich domain), on five different mouse models of HD using a chimeric adeno-associated viral 2/1 vector (AAV2/1) with a modified cytomegalovirus enhancer/chicken beta-actin promoter. Interestingly, contradictory results were obtained with AAV-V(L)12.3 treatment depending on the model used, with beneficial effects observed in a lentiviral mouse HD model, no effects detected in the YAC128 mouse model, and an increase in the severity of the phenotype of the R6/2 model. On the other hand, AAV-Happ1 treatment strongly ameliorated the neuropathology (e.g., the enlargement of the lateral ventricles) and behavioral deficits of all five HD mouse models (i.e., the lentiviral model, the R6/2 and N171–82Q truncated transgenic models, as well as the YAC128 and the BAC full-length transgenic models), and significantly increased the survival of N171–82Q HD mice [116]. Because increasing the turnover of mutant huntigtin using AAV-Happ1 gene therapy was an effective treatment in diverse rodent models of HD including the YAC128 mice, the use of antihuntingtin intrabodies might be a novel therapeutic approach for HD.

Final Conclusions

The generation of YAC128 HD transgenic mice that express the full-length HD gene with 128 CAG repeats [12] has greatly improved our understanding of the mechanisms underlying this neurodegenerative disorder in humans. The deterioration of their motor function closely resembles the human condition, following a byphasic pattern over the course of their life-span [12]. Moreover, these transgenic mice also show cognitive impairments [20] and a depressive-like behavior [22] that precedes, as in humans, the overt motor symptoms. Finally, and contrary to what has been reported in other HD transgenic mouse models, the YAC128 HD mice develop striatal neuronal loss [12], the neuropathological hallmark of HD. These characteristics and the phenotypic uniformity with low interanimal variability make this transgenic mouse model a unique tool for the elucidation of several mechanisms that contribute to HD pathogenesis as well as the screening of novel neuroprotective therapeutic approaches for the treatment of this neurodegenerative disorder. In agreement, studies in YAC128 mice have greatly contributed to our understanding of the excitotoxic [46] and apoptotic [17] pathways that are activated in the HD brain and that can be targeted for possible therapeutic interventions. Moreover, these HD mice have also been invaluable for the testing of innovative therapeutic approaches such as the use of intrabodies directed against mutant huntingtin [116]. This new treatment strategy in particular was shown to be successful in mitigating the behavioral and neuropathological deficits not only in the YAC128 model but also in several other HD transgenic mice [116], thus making the development of intrabodies for the treatment of the human condition a novel avenue in HD research. To conclude, the use of the YAC128 HD transgenic mouse model will continue to be an invaluable tool not only in the elucidation of the neurobiology of HD, but also in the ultimate quest to find a cure for this devastating neurodegenerative disorder.


The author acknowledges postdoctoral funding from the Natural Sciences and Engineering Research Council of Canada. The author also wishes to thank Jessica M. Simpson for critically reading this manuscript.

Conflict of Interest

The authors has no conflict of interest.