Striosome-matrix pathology and motor deficits in the YAC128 mouse model of Huntington's disease

Huntington's disease is characterized by striatal degeneration and progressive motor deficits. To examine striatal compartment-specific pathology and its relation to motor symptoms, we used immunohistochemistry to identify and measure the striosomes and matrix of 7–13-month-old YAC128 and wild type (WT) mice that were previously tested on motor tasks. Compared to WTs, 13-month-old YAC128s showed volume shrinkage in striosomes, and cell loss in both compartments. The percent cell loss was greater in striosomes than matrix. Striosome volume and cell loss was greatest in the dorsolateral striatum. YAC128 rotarod and balance beam deficits preceded volume and cell loss. At 13 months, YAC128 balance beam slips and striosome cell number were inversely correlated. The results show that pathology in older YAC128s manifests as an abnormal striosome to matrix ratio and suggest that this imbalance can contribute to some motor symptoms.     

Beneficial effects of rolipram in the R6/2 mouse model of Huntington's disease

We have previously showed that rolipram, a phosphodiesterase type IV inhibitor, displays a neuroprotective effect in a rat quinolinic acid model of HD [DeMarch Z., Giampa C., Patassini S., Martorana A., Bernardi G. and Fusco F.R., (2007) Beneficial effects of rolipram in a quinolinic acid model of striatal excitotoxicity. Neurobiol. Dis. 25:266–273.]. In this study, we sought to determine if rolipram exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD [Mangiarini L., Sathasivam K., Seller M., Cozens B., Harper A., Hetherington C., Lawton M., Trottier Y., Lehrach H., Davies S.W. and Bates G.P. (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell. 87:493–506]. Transgenic mice were treated with rolipram 1.5 mg/kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that rolipram-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as brain volume, striatal atrophy, size and morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. Rolipram was effective in increasing significantly the levels of activated CREB and of BDNF the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that rolipram could be considered as a valid therapeutic approach for HD.     

Reduced activity of cortico-striatal fibres in the R6/2 mouse model of Huntington's disease

We have used the R6/2 mice to study cortico-striatal glutamatergic transmission by microdialysis in freely moving mice. Basal extracellular striatal glutamate concentrations were lower in R6/2 mice at 12 weeks of age, but not at 6 weeks of age, when neurological symptoms start to develop. In contrast, K-induced glutamate release was blunted in the striatum of R6/2 mice at both 6 and 12 weeks of age as compared with age-matched controls. We also found a substantial reduction in striatal pro-BDNF (brain derived neurotrophic factor) levels associated with no changes in the mature form of BDNF, as assessed by immunoblotting, in 12-week-old R6/2 mice, suggesting a reduced turnover rate of BDNF in the striatum of these mice. These data support the hypothesis of a cortico-striatal dysfunction in Huntington's disease.     

Abnormal sensitivity to cannabinoid receptor stimulation might contribute to altered gamma-aminobutyric acid transmission in the striatum of R6/2 Huntington's disease mice.

BACKGROUND: One of the earliest neurochemical alterations observed in both Huntington's disease (HD) patients and HD animal models is the dysregulation of the endocannabinoid system, an alteration that precedes the development of identifiable striatal neuropathology. How this alteration impacts striatal synaptic transmission is unknown. METHODS: We measured the effects of cannabinoid receptor stimulation on gamma-aminobutyric acid (GABA)-ergic synaptic currents recorded from striatal neurons of R6/2 HD mice in the early phase of their disease. RESULTS: The sensitivity of striatal GABA synapses to cannabinoid receptor stimulation is severely impaired in R6/2 HD mice. In particular, whereas in control animals activation of cannabinoid CB1 receptors results in a significant inhibition of both evoked and spontaneous GABA-mediated synaptic events by a presynaptic mechanism, in R6/2 mice this treatment fails to reduce GABA currents but causes, in contrast, a slight increase of spontaneous inhibitory postsynaptic currents (sIPSCs). CONCLUSIONS: Experimental HD was also associated with enhanced frequency of sIPSCs, a result consistent with the conclusion that loss of cannabinoid-mediated control of GABA transmission might contribute to hyperactivity of GABA synapses in the striatum of HD mice. Accordingly, spontaneous excitatory postsynaptic currents, which were not upregulated in R6/2 mice, were still sensitive to cannabinoid receptor stimulation.     

Differential changes in striatal projection neurons in R6/2 transgenic mice for Huntington's disease

In early adult-onset Huntington's disease (HD), enkephalinergic striatopallidal projection neurons show preferential loss, reduced preproenkephalin (PPE) expression in surviving striatopallidal neurons, and loss of fibers in their projection target area. We examined PPE and PPT (preprotachykinin) gene expression in striatal projection neurons and in striatal projection fibers immunoreactive for the PPE product enkephalin (ENK) and the PPT product substance P (SP) in a transgenic HD model, the R6/2 mouse, to see if changes occur in these neuron types similar to those seen in early adult-onset HD. The results show that PPE mRNA level, the number of striatal neurons expressing PPE, and the staining intensity of fibers immunoreactive for ENK in the pallidum were all decreased. By contrast, the SP-containing striatal projection systems to the pallidum and substantia nigra were relatively normal in R6/2 mice. The selective reduction in striatal PPE in R6/2 mice is reminiscent of adult-onset HD, but the preservation of the striatonigral projection system is not. Thus, R6/2 mice do not strictly mimic adult-onset HD in their striatal pathology.     

Increased metabolism in the R6/2 mouse model of Huntington's disease

Huntington's disease (HD) is a hereditary disorder characterized by personality changes, chorea, dementia and weight loss. The cause of this weight loss is unknown. The aim of this study was to examine body weight changes and weight-regulating factors in HD using the R6/2 mouse model as a tool. We found that R6/2 mice started losing weight at 9 weeks of age. Total locomotor activity was unaltered and caloric intake was not decreased until 11 weeks of age, which led us to hypothesize that increased metabolism might underlie the weight loss. Indeed, oxygen consumption in R6/2 mice was elevated from 6 weeks of age, indicative of an increased metabolism. Several organ systems that regulate weight and metabolism, including the hypothalamus, the stomach and adipose tissue displayed abnormalities in R6/2 mice. Together, these data demonstrate that weight loss in R6/2 mice is associated with increased metabolism and changes in several weight-regulating factors.     

Cardiac dysfunction in the R6/2 mouse model of Huntington's disease

Recent evidence suggests that mutant huntingtin protein-induced energetic perturbations contribute to neuronal dysfunction in Huntington's disease (HD). Given the ubiquitous expression of huntingtin, other cell types with high energetic burden may be at risk for HD-related dysfunction. Early-onset cardiovascular disease is the second leading cause of death in HD patients; a direct role for mutant huntingtin in this phenomenon remains unevaluated. Here we tested the hypothesis that expression of mutant huntingtin is sufficient to induce cardiac dysfunction, using a well-described transgenic model of HD (line R6/2). R6/2 mice developed cardiac dysfunction by 8 weeks of age, progressing to severe failure at 12 weeks, assessed by echocardiography. Limited evidence of cardiac remodeling (e.g. hypertrophy, fibrosis, apoptosis, beta(1) adrenergic receptor downregulation) was observed. Immunogold electron microscopy demonstrated significant elevations in nuclear and mitochondrial polyglutamine presence in the R6/2 myocyte. Significant alterations in mitochondrial ultrastructure were seen, consistent with metabolic stress. Increased cardiac lysine acetylation and protein nitration were observed and were each significantly associated with impairments in cardiac performance. These data demonstrate that mutant huntingtin expression has potent cardiotoxic effects; cardiac failure may be a significant complication of this important experimental model of HD. Investigation of the potential cardiotropic effects of mutant huntingtin in humans may be warranted.     

Limbic neurogenesis/plasticity in the R6/2 mouse model of Huntington's disease

Huntington's disease is an inherited neurodegenerative condition characterized by movement disorders, and mood and cognitive disturbance. Mammalian neurogenesis persists into adulthood in the subventricular zone and dentate gyrus of the hippocampus. Neurogenesis is abnormal in the dentate gyrus in the R6/2 transgenic mouse model of Huntington's disease. We have now found that the number of immature neurons (doublecortin-positive cells) is markedly reduced in the piriform and insular cortex but not in the temporal germinal layer or caudal subventricular zone of R6/2 mice. Furthermore, numbers of such cells were unaltered in response to seizures in both wild-type and R6/2 mice. These results support the possibility that impaired neurogenesis and/or plasticity could contribute to cognitive and psychiatric impairments in Huntington's disease.     

Reduction of GnRH and infertility in the R6/2 mouse model of Huntington's disease

Reductions in testosterone and luteinizing hormone levels and reduced sexual functions have been reported in Huntington's disease (HD) patients. Atrophy of the reproductive organs and loss of fertility have also been observed in the R6/2 mouse, which is currently the most studied transgenic model of HD. In an effort to define the cause of infertility we studied the expression of gonadotropin-releasing hormone (GnRH) in the medial septum, diagonal band of Broca and hypothalamus of R6/2 male mice during sexual maturation. We found a progressive reduction in the numbers of GnRH-immunoreactive neurons in the analysed brain areas of R6/2 mice starting at 5 weeks of age and becoming statistically significant with only 10% of the neurons remaining by 9 weeks of age. Atrophy of testes and seminal vesicles combined with a significant reduction in serum and testicular testosterone levels were detected in 12-week-old R6/2mice. These results suggest that infertility in the R6/2 males is due either to death of GnRH neurons or to a reduction in GnRH expression leading to a downstream impairment of the gonadotropic hormones. Gonadotropic hormone replacement did not mitigate weight loss or restore motor function in R6/2 males.     

Tiagabine is neuroprotective in the N171-82Q and R6/2 mouse models of Huntington's disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by chorea, incoordination, and shortened life-span, and by huntingtin inclusions and neurodegeneration. We previously screened the 1040 FDA-approved compounds from the NINDS compound library and found that a compound, nipecotic acid, significantly reduced mutant huntingtin aggregations and blocked cell toxicity in an inducible cell model of HD. Because nipecotic acid does not cross the blood–brain barrier (BBB), we studied its analogue, tiagabine, which is able to cross the BBB, in both N171-82Q and R6/2 transgenic mouse models of HD. Tiagabine was administered intraperitoneally at 2 and 5 mg/kg daily in HD mice. We found that tiagabine extended survival, improved motor performance, and attenuated brain atrophy and neurodegeneration in N171-82Q HD mice. These beneficial effects were further confirmed in R6/2 HD mice. The levels of tiagabine at effective doses in mouse serum are comparable to the levels in human patients treated with tiagabine. These results suggest that tiagabine may have beneficial effects in the treatment of HD. Because tiagabine is an FDA-approved drug, it may be a promising candidate for future clinical trials for the treatment of HD.     

Reduced hippocampal neurogenesis in R6/2 transgenic Huntington's disease mice

We investigated whether cell proliferation and neurogenesis are altered in R6/2 transgenic Huntington's disease mice. Using bromodeoxyuridine (BrdU), we found a progressive decrease in the number of proliferating cells in the dentate gyrus of R6/2 mice. This reduction was detected in pre-symptomatic mice, and by 11.5 weeks, R6/2 mice had 66% fewer newly born cells in the hippocampus. The results were confirmed by immunohistochemistry for the cell cycle markers Ki-67 and proliferating cell nuclear antigen (PCNA). We did not observe changes in cell proliferation in the R6/2 subventricular zone, indicating that the decrease in cell proliferation is specific for the hippocampus. This decrease corresponded to a reduction in actual hippocampal neurogenesis as assessed by double immunostaining for BrdU and the neuronal marker neuronal nuclei (NeuN) and by immunohistochemistry for the neuroblast marker doublecortin. Reduced hippocampal neurogenesis may be a novel neuropathological feature in R6/2 mice that could be assessed when evaluating potential therapies.     

The effects of intranigral GABA and dynorphin A injections on striatal dopamine and GABA release: Evidence that dopamine provides inhibitory regulation of striatal GABA neurons via D2 receptors

The effects of injections of γ-aminobutyric acid (GABA) and dynorphin A into the substantia nigra, pars reticulata on the levels of extracellular dopamine (DA) and GABA in the ipsilateral striatum of halothane-anaesthetized rats were studied using microdialysis. The effects of intranigral injections of substance P and neurokinin A were also studied. Intranigral GABA (300 nmol) or dynorphin A (0.5 nmol) injections produced a simultaneous decrease in DA and increase in GABA levels, while intranigral substance P (0.07 nmol) or neurokinin A (0.09 nmol) injections produced an increase in DA but had no effect on GABA levels. DA agnonists, apomorphine (D₁/D₂), SKF 38393 (D₁) and pergolide (D₂) were applied locally by perfusing them through the microdialysis probe, each at a concentration of 10⁻⁵ M. All 3 agonists decreased the levels of DA in the striatum. However, while apomorphine and SKF 38393 increased, pergolide decreased the levels of GABA in the striatum. The increase in striatal GABA produced by intranigral injections of GABA (300 nmol) was reversed by local perfusion with pergolide (10⁻⁵ M), but was not reversed by local perfusion with SKF 38393 (10⁻⁵ M). These findings suggest that D₁ and D₂ receptors differentially regulate striatal GABA release, and are stimulatory and inhibitory, respectively. Furthermore, it is suggested that nigrostriatal DA functions as an inhibitory modulator of striatal GABA neurons, acting via D₂ receptors.     

Reduced expression of PSA-NCAM in the hippocampus and piriform cortex of the R6/1 and R6/2 mouse models of Huntington's disease

Cognitive deficits and impaired olfactory function are observed in early stages of Huntington's disease (HD). The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is strongly associated with plastic events in the brain. During adulthood, it is most abundantly expressed in the hippocampus and the piriform cortex, which are involved in cognition and olfaction, respectively. We show that the numbers of PSA-NCAM-positive cells in the hippocampus and piriform cortex are dramatically reduced in the R6/1 and the R6/2 mouse models of HD. We hypothesize that the decrease in NCAM polysialylation reflects an impaired plasticity and might underlie some of the early symptoms in HD.     

Increased thirst and drinking in Huntington's disease and the R6/2 mouse

While Huntington's disease (HD) is a condition that primarily involves the basal ganglia, there is evidence to suggest that the hypothalamus is also affected. Because the osmoreceptors regulating thirst are situated in the circumventricular region of the hypothalamus, we were interested in whether altered thirst is a part of the HD phenotype. We used the LABORAS behavioural monitoring system and water consumption to show that drinking behaviour was abnormal in R6/2 mice. By 10 weeks of age, R6/2 mice spent significantly more time drinking and drank a greater volume than their wild-type (WT) littermates. The numbers of immunoreactive vasopressin neurons in the paraventricular nucleus (PVN) of the hypothalamus in R6/2 mice were significantly decreased from 8 weeks of age, suggesting that the change in drinking behaviour may be the result of hypothalamic dysfunction. We gave a xerostomia (dry mouth) questionnaire to HD patients and control subjects, and also measured their urine osmolality and serum vasopressin. The mean total xerostomia score was significantly higher in HD patients than in controls, indicating greater thirst in HD patients. Urine osmolality was unaffected in HD patients up to clinical stage III, and none of the patients had diabetes. However, serum vasopressin was increased, suggesting a dysregulation in the control of hypothalamic vasopressin release. A dry mouth can affect taste, mastication and swallowing, all of which may contribute to the significant weight loss seen in both HD patients and R6/2 mice, as can dehydration. We suggest that increased thirst may be an important and clinically relevant biomarker for the study of disease progression in HD.     

Phosphodiesterase 10 inhibition reduces striatal excitotoxicity in the quinolinic acid model of Huntington's disease

Decreased activity of cAMP responsive element-binding protein (CREB) is thought to contribute to the death of striatal medium spiny neurons in Huntington's disease (HD). Therefore, therapies that increase levels of activated CREB, may be effective in fighting neurodegeneration in HD. In this study, we sought to determine whether the phosphodiesterase type 10 (PDE10A) inhibitor TP10 exerts a neuroprotective effect in an excitotoxic model of HD. Rats were surgically administered with quinolinic acid into striatum and subsequently treated with TP10 daily for two or eight weeks. After 2 weeks of TP10 treatment, striatal lesion size was 52% smaller and the surviving cell number was several times higher than in the vehicle-treated group. These beneficial effects of TP10 were maintained through 8 weeks. TP10 treatment also increased significantly the levels of activated CREB in the striatal spiny neurons, which is hypothesized to be a contributing mechanism for the neuroprotective effect. Our findings suggest PDE10A inhibition as a novel neuroprotective approach to the treatment of HD and confirm the importance of phosphodiesterase inhibition in fighting the disease.     

Reduced expression of conditioned fear in the R6/2 mouse model of Huntington's disease is related to abnormal activity in prelimbic cortex

Prefrontal cortex (PFC) dysfunction is common in patients with Huntington's disease (HD), a dominantly inherited neurological disorder, and has been linked to cognitive disruption. We previously reported alterations in neuronal firing patterns recorded from PFC of the R6/2 mouse model of HD. To determine if PFC dysfunction results in behavioral impairments, we evaluated performance of wild-type (WT) and R6/2 mice in a fear conditioning and extinction behavioral task. Fear conditioning and extinction retrieval were similar in both genotypes, but R6/2s exhibited less fear during extinction by freezing less than WTs. A fear reinstatement test after extinction retrieval indicated that faster extinction was not due to poor memory for conditioning. During initial extinction and extinction retrieval training, neuronal activity was recorded from prelimbic (PL) cortex, a subregion of PFC known to be important for fear expression. In WTs, a large number of neurons were activated by the conditioned stimulus during initial extinction and this activation was significantly impaired in R6/2s. Notably, there was no genotype difference in PFC activity during extinction retrieval. Thus, altered extinction is likely a result of reduced fear expression due to impairments in PL activation. Collectively, our results suggest that PFC dysfunction may play a key role in R6/2 cognitive impairments.     

Progressive dysfunction of the cholesterol biosynthesis pathway in the R6/2 mouse model of Huntington's disease

We have recently reported significantly reduced levels of the mRNA of genes critical for the cholesterol biosynthesis pathway in the brains of mice and patients with Huntington's disease (HD), which are indicative of a biological dysfunction. We here show that the brains of R6/2 transgenic mice have progressively decreasing levels of the cholesterol precursors, lathosterol and lanosterol, and declining 3-hydroxy-3-methylglutaryl coenzyme A reductase activity starting from pre-symptomatic stages. We also show that, despite the progressive reduction of brain cholesterol biosynthesis, steady-state levels of total cholesterol remain constant, thus suggesting that compensatory mechanisms are in operation. These in vivo findings indicate a consistent and progressive reduction in the activity of the cholesterol biosynthesis pathway in HD brain. The defect occurs early in these mice and generates lower levels of newly synthesized cholesterol and its intermediates, which may affect different aspects of the disease.     

Impaired glucose tolerance in the R6/1 transgenic mouse model of Huntington's disease

Previous reports have highlighted a possible link between Huntington's disease (HD) and diabetes mellitus (DM), but the association has not been characterised in detail. A transgenic mouse model for HD, the R6/2 mouse, also develops diabetes. In the present study, we examined the R6/1 mouse, which carries a shorter CAG repeat than the R6/2 mouse, and found that, although not diabetic, the mice showed several signs of impaired glucose tolerance. First, following i.p. glucose injection, the blood glucose concentration was approximately 30% higher in young R6/1 mice (10 weeks) compared to wild-type mice (P = 0.004). In older mice (38 weeks), glucose tolerance was further impaired in both R6/1 and wild-type animals. Second, during glucose challenge, the R6/1 mice reached higher plasma insulin levels than wild-type mice, but the peripheral insulin sensitivity was normal as measured by injection of human or mouse insulin or when evaluated by the quantitative insulin sensitivity check index (QUICKI). Third, the beta cell volume was 17% and 39% smaller at 10 and 38 weeks of age, respectively, compared to age-matched wild-type littermates and the reduction was not caused by apoptosis at either age. Finally, we demonstrated the presence of the HD gene product, huntingtin (htt), in both alpha- and beta-cells in R6/1 islets of Langerhans. Since pancreatic beta cells and neurons share several common traits, clarification of the mechanism associating neurodegenerative diseases with diabetes might improve our understanding of the pathogenic events leading to both groups of diseases.     

CAG repeat lengths ≥ 335 attenuate the phenotype in the R6/2 Huntington's disease transgenic mouse

With spontaneous elongation of the CAG repeat in the R6/2 transgene to ≥ 335, resulting in a transgene protein too large for passive entry into nuclei via the nuclear pore, we observed an abrupt increase in lifespan to > 20 weeks, compared to the 12 weeks common in R6/2 mice with 150 repeats. In the ≥ 335 CAG mice, large ubiquitinated aggregates of mutant protein were common in neuronal dendrites and perikaryal cytoplasm, but intranuclear aggregates were small and infrequent. Message and protein for the ≥ 335 CAG transgene were reduced to one-third that in 150 CAG R6/2 mice. Neurological and neurochemical abnormalities were delayed in onset and less severe than in 150 CAG R6/2 mice. These findings suggest that polyQ length and pathogenicity in Huntington's disease may not be linearly related, and pathogenicity may be less severe with extreme repeats. Both diminished mutant protein and reduced nuclear entry may contribute to phenotype attenuation.     

Oral uridine pro-drug PN401 is neuroprotective in the R6/2 and N171-82Q mouse models of Huntington's disease

Previously, uridine pro-drug 2',3',5'-tri-O-acetyluridine (PN401) was shown to be protective in the mitochondrial complex II inhibitor 3-nitropropionic acid model of Huntington's disease (HD). In this study, PN401 increased survival and improved motor function on the rotarod in both R6/2 and N171-82Q polyglutamine repeat mouse models of HD. PN401 significantly decreased neurodegeneration in both the piriform cortex and striatum although PN401 decreased huntingtin protein aggregates only in the striatum. Cortical and striatal brain-derived neurotrophic factor (BDNF) protein levels were reduced in the +/- compared to the -/- N171-82Q mice and PN401 treatment significantly increased cortical BDNF in both +/- and -/- mice, but PN401 did not affect striatal BDNF. These results suggest that PN401 may have beneficial effects in the treatment of neurodegenerative diseases such as HD.