Changes in GAD67 mRNA expression evidenced by in situ hybridization in the brain of R6/2 transgenic mice.

Huntington's disease is an autosomal dominant disorder with degeneration of medium size striatal neurones. As the disease evolves, other neuronal populations are also progressively affected. A transgenic mouse model of the disease (R6/2) that expresses exon 1 of the human Huntington gene with approximately 150 CAG repeats has been developed, but GABA concentrations are reported to be normal in the striatum of these animals. In the present study, we analysed the status of GABAergic systems by means of glutamic acid decarboxylase (GAD)67 mRNA in situ hybridization in the brain of R6/2 transgenic mice and wild-type littermates. We show that GAD67 expression is normal in the striatum, cerebellum and septum but decreased in the frontal cortex, parietal cortex, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata of R6/2 mice. These data, which may, in part, account for the behavioural changes seen in these animals, indicate that at 12.5 weeks of age the pathological features seen in the mice differ from those seen in humans with Huntington's disease.

Loss of cortical and thalamic neuronal tenascin-C expression in a transgenic mouse expressing exon 1 of the human Huntington disease gene.

A transgenic mouse containing the first exon of the human Huntington's disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntington's disease (HD). The present study has found that expression of the extracellular matrix glycoprotein tenascin-C appears to be unaffected in astroglial cells in wild-type and R6/2 transgenic mice that express the mutant huntingtin protein but that it is conspicuously absent in two neuronal populations within the cerebral cortex and thalamus of the R6/2 mice. Loss of tenascin-C expression begins between the fourth and eighth postnatal weeks, coincidental with the onset of abnormal behavioral phenotype and the appearance of intranuclear inclusion bodies and neuropil aggregates. By 12 weeks, R6/2 mice exhibit a complete absence of tenascin-C neuronal immunolabeling, a disappearance of cRNA probe-positive neurons across discrete cytoarchitectonic regions of the dorsal thalamus (e.g., the ventromedial, parafascicular, lateral posterior, and posterior thalamic groups) and frontal cortex, and an accompanying thalamic astrogliosis. The loss of neuronal tenascin-C expression includes structures that are known to send converging excitatory axonal projections to the caudate-putamen, the structure that is most at risk for neurodegeneration in HD. Altered neuronal expression of tenascin-C in R6/2 mice implicates altered transcriptional activities of the mutant huntingtin protein. The abnormal biochemistry and possibly abnormal activity of thalamostriate and corticostriate projection neurons may also affect abnormal neuronal activities in their primary connectional target, the neostriatum, which is severely compromised in HD.

Amyloid-like inclusions in Huntington's disease.

Huntington's disease is a progressive, autosomal dominantly inherited, neurodegenerative disease that is characterized by involuntary movements (chorea), cognitive decline and psychiatric manifestations. This is one of a number of late-onset neurodegenerative disorders caused by expanded glutamine repeats, with a likely similar biochemical basis. Immunohistochemical studies on Huntington's disease tissue, using antibodies raised to the N-terminal region of huntingtin (adjacent to the repeat) and ubiquitin, have recently identified neuronal inclusions within densely stained neuronal nuclei, peri-nuclear and within dystrophic neuritic processes. However, the functional significance of inclusions is unknown. It has been suggested that the disease-causing mechanism in Huntington's disease (and the other polyglutamine disorders) is the ability of polyglutamine to undergo a conformational change that can lead to the formation of very stable anti-parallel beta-sheets; more specifically, amyloid structures. We examined, using Congo Red staining and both polarizing and confocal microscopy, post mortem human brain tissue from five Huntington's disease cases, two Alzheimer's disease cases and two normal controls. Brains from five transgenic mice (R6/2)(12) expressing exon 1 of the human huntingtin gene with expanded polyglutamine, and five littermate controls, were also examined by the same techniques. We have shown that some inclusions in Huntington's disease brain tissue possess an amyloid-like structure, suggesting parallels with other amyloid-associated diseases such as Alzheimer's and prion diseases.

Abnormal synaptic plasticity and impaired spatial cognition in mice transgenic for exon 1 of the human Huntington's disease mutation.

Huntington's disease (HD) is an autosomal dominant progressive and fatal neurodegenerative brain disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the gene. Presymptomatic Huntington's disease patients often exhibit cognitive deficits before the onset of classical symptoms. To investigate the possibility that changes in synaptic plasticity might underlie cognitive impairment in HD, we examined hippocampal synaptic plasticity and spatial cognition in a transgenic mouse (R6/2 line) expressing exon 1 of the human Huntington's disease gene containing an expanded CAG repeat. This mouse exhibits a progressive and fatal neurological phenotype that resembles Huntington's disease. We report that R6/2 mice show marked alterations in synaptic plasticity at both CA1 and dentate granule cell synapses, and impaired spatial cognitive performance in the Morris water maze. The changes in hippocampal plasticity were age dependent, appearing at CA1 synapses several weeks before they were observed in the dentate gyrus. Deficits in synaptic plasticity at CA1 synapses occurred before an overt phenotype. This suggests that altered synaptic plasticity contributes to the pre-symptomatic changes in cognition reported in human carriers of the Huntington' disease gene. The temporal and regional changes in synaptic plasticity within the hippocampus mirror the appearance of neuronal intranuclear inclusions, suggesting a relationship between polyglutamine aggregation and dysfunction.

Nonapoptotic neurodegeneration in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal inherited neurodegenerative disorder characterized by personality changes, motor impairment, and subcortical dementia. HD is one of a number of diseases caused by expression of an expanded polyglutamine repeat. We have developed several lines of mice that are transgenic for exon 1 of the HD gene containing an expanded CAG sequence. These mice exhibit a defined neurological phenotype along with neuronal changes that are pathognomonic for the disease. We have previously observed the appearance of neuronal intranuclear inclusions, but did not find evidence for neurodegeneration. In this study, we report that all lines of these mice develop a late onset neurodegeneration within the anterior cingulate cortex, dorsal striatum, and of the Purkinje neurons of the cerebellum. Dying neurons characteristically exhibit neuronal intranuclear inclusions, condensation of both the cytoplasm and nucleus, and ruffling of the plasma membrane while maintaining ultrastructural preservation of cellular organelles. These cells do not develop blebbing of the nucleus or cytoplasm, apoptotic bodies, or fragmentation of DNA. Neuronal death occurs over a period of weeks not hours. We also find degenerating cells of similar appearance within these same regions in brains of patients who had died with HD. We therefore suggest that the mechanism of neuronal cell death in both HD and a transgenic mouse model of HD is neither by apoptosis nor by necrosis.

Mitochondrial dysfunction and free radical damage in the Huntington R6/2 transgenic mouse.

Huntington's disease is a progressive neurodegenerative disease caused by an abnormally expanded (>36) CAG repeat within the ITI5 gene encoding a widely expressed 349-kd protein, huntingtin. The medium spiny neurons of the caudate preferentially degenerate in Huntington's disease, with the presence of neuronal intranuclear inclusions. Excitotoxicity is thought to be important in the pathogenesis of Huntington's disease; the recently described mitochondrial respiratory chain and aconitase defects in Huntington's disease brain are consistent with this hypothesis. A transgenic mouse model (R6/2) of Huntington's disease develops a movement disorder, muscle wasting, and premature death at about 14 to 16 weeks. Selective neuronal death in these mice is not seen until 14 weeks. Biochemical analysis of R6/2 mouse brain at 12 weeks demonstrated a significant reduction in aconitase and mitochondrial complex IV activities in the striatum and a decrease in complex IV activity in the cerebral cortex. Increased immunostaining for inducible nitric oxide synthase and nitrotyrosine was seen in the transgenic mouse model but not control mouse brains. These results extend the parallels between Huntington's disease and the transgenic mouse model to biochemical events and suggest complex IV deficiency and elevated nitric oxide and superoxide radical generation precede neuronal death in the R6/2 mouse and contribute to pathogenesis.

Transgenic models of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion. A mouse model of this disease has been generated by the introduction of exon 1 of the human HD gene carrying highly expanded CAG repeats into the mouse germ line (R6 lines). Transgenic mice develop a progressive neurological phenotype with a movement disorder and weight loss similar to that in HD. We have previously identified neuronal inclusions in the brains of these mice that have subsequently been established as the pathological hallmark of polyglutamine disease. Inclusions are present before symptoms, which in turn occur long before any selective neuronal cell death can be identified. We have extended the search for inclusions to skeletal muscle, which, like brain, contains terminally differentiated cells. We have conducted an investigation into the skeletal muscle atrophy that occurs in the R6 lines, (i) to provide possible insights into the muscle bulk loss observed in HD patients, and (ii) to conduct a parallel analysis into the consequence of inclusion formation to that being performed in brain. The identification of inclusions in skeletal muscle might be additionally useful in monitoring the ability of drugs to prevent inclusion formation in vivo.

From neuronal inclusions to neurodegeneration: neuropathological investigation of a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease caused by the expansion of a polyglutamine repeat sequence within a novel protein. Recent work has shown that abnormal intranuclear inclusions of aggregated mutant protein within neurons is a characteristic feature shared by HD and several other diseases involving glutamine repeat expansion. This suggests that in each of the these disorders the affected nerve cells degenerate as a result of these abnormal inclusions. A transgenic mouse model of HD has been generated by introducing exon 1 of the HD gene containing a highly expanded CAG sequence into the mouse germline. These mice develop widespread neuronal intranuclear inclusions and neurodegeneration specifically within those areas of the brain known to degenerate in HD. We have investigated the sequence of pathological changes that occur after the formation of nuclear inclusions and that precede neuronal cell death in these cells. Although the relation between inclusion formation and neurodegeneration has recently been questioned, a full characterization of the pathways linking protein aggregation and cell death will resolve some of these controversies and will additionally provide new targets for potential therapies.

Altered neurotransmitter receptor expression in transgenic mouse models of Huntington's disease.

Alterations in neurotransmitter receptors are a pathological hallmark of the neurodegeneration seen in Huntington's disease (HD). However, the significance of these alterations has been uncertain, possibly reflecting simply the loss of brain cells. It is not known for certain whether the alteration of neurotransmitter receptors occurs before the onset of symptoms in human HD. Recently we developed transgenic mice that contain a portion of a human HD gene and develop a progressive abnormal neurological phenotype. Neurotransmitter receptors that are altered in HD (receptors for glutamate, dopamine, acetylcholine and adenosine) are decreased in the brain transgenic mice, in some cases before the onset of behavioural or motor symptoms. In transgenic mice, neurotransmitter receptor alterations occur before neuronal death. Further, receptor alterations are selective in that certain receptors, namely N-methyl-D-aspartate and gamma-aminobutyric acid receptors, are unaltered. Finally, receptor decreases are preceded by selective decreases in the corresponding mRNA species, suggesting the altered transcription of specific genes. These results suggest that (i) receptor decreases precede, and therefore might contribute to, the development of clinical symptoms, and (ii) altered transcription of specific genes might be a key pathological mechanism in HD.

Ultrastructural localization and progressive formation of neuropil aggregates in Huntington's disease transgenic mice.

How aggregates of polyglutamine proteins are involved in the neurological symptoms of glutamine repeat diseases is unknown. We show that huntingtin aggregates are present in the neuronal processes of transgenic mice that express exon 1 of the Huntington's disease (HD) gene. Unlike aggregates in the nucleus, these neuropil aggregates are usually smaller and are not ubiquitinated. Electron microscopy reveals many neuropil aggregates in axons and axon terminals. Huntingtin aggregates in the axon terminal are co-localized with some synaptic vesicles, implying that they may affect synaptic transmission and neuronal communication. The formation of neuropil aggregates is highly correlated with the development of neurological symptoms. The present study raises the possibility that neuropil aggregates may cause a dysfunction in neuronal communication and con-tribute to the neurological symptoms of HD.

Formation of polyglutamine inclusions in non-CNS tissue.

Huntington's disease (HD) is one of a class of inherited progressive neurodegenerative disorders that are caused by a CAG/polyglutamine repeat expansion. We have previously generated mice that are transgenic for exon 1 of the HD gene carrying highly expanded CAG repeats which develop a progressive movement disorder and weight loss with similarities to HD. Neuronal inclusions composed of the exon 1 protein and ubiquitin are present in specific brain regions prior to onset of the phenotype, which in turn occurs long before specific neurodegeneration can be detected. In this report we have extended the search for polyglutamine inclusions to non-neuronal tissues. Outside the central nervous system (CNS), inclusions were identified in a variety of post-mitotic cells. This is consistent with a concentration-dependent nucleation and aggregation model of inclusion formation and indicates that brain-specific factors are not necessary for this process. To possibly gain insights into the wasting that is observed in the human disease, we have conducted a detailed analysis of the timing and progression of inclusion formation in skeletal muscle and an investigation into the cause of the severe muscle atrophy that occurs in the mouse model. The formation of inclusions in non-CNS tissues will be particularly useful with respect to in vivo monitoring of pharmaceutical agents selected for their ability to prevent polyglutamine aggregation in vitro, without the requirement that the agent can cross the blood-brain barrier in the first instance.

Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation.

Transgenic mice expressing exon 1 of the human Huntington's disease (HD) gene carrying a 141-157 CAG repeat (line R6/2) develop a progressive neurological phenotype with motor symptoms resembling those seen in HD. We have characterized the motor deficits in R6/2 mice using a battery of behavioral tests selected to measure motor aspects of swimming, fore- and hindlimb coordination, balance, and sensorimotor gating [swimming tank, rotarod, raised beam, fore- and hindpaw footprinting, and acoustic startle/prepulse inhibition (PPI)]. Behavioral testing was performed on female hemizygotic R6/2 transgenic mice (n = 9) and female wild-type littermates (n = 22) between 5 and 14 weeks of age. Transgenic mice did not show an overt behavioral phenotype until around 8 weeks of age. However, as early as 5-6 weeks of age they had significant difficulty swimming, traversing the narrowest square (5 mm) raised beam, and maintaining balance on the rotarod at rotation speeds of 33-44 rpm. Furthermore, they showed significant impairment in prepulse inhibition (an impairment also seen in patients with HD). Between 8 and 15 weeks, R6/2 transgenic mice showed a progressive deterioration in performance on all of the motor tests. Thus R6/2 mice show measurable deficits in motor behavior that begin subtly and increase progressively until death. Our data support the use of R6/2 mice as a model of HD and indicate that they may be useful for evaluating therapeutic strategies for HD, particularly those aimed at reducing the severity of motor symptoms or slowing the course of the disease.

Brain neurotransmitter deficits in mice transgenic for the Huntington's disease mutation.

Huntington's disease (HD) is associated with an expansion in the CAG repeat sequence of a gene on chromosome 4, resulting in a neurodegenerative process particularly affecting the striatum and with profound but selective changes in content of various neurotransmitters. Recently, transgenic mice expressing a fragment of the human HD gene containing a large CAG expansion have been generated; these mice exhibit a progressive neurological phenotype that includes motor disturbances, as well as neuronal deficits. To investigate their underlying neurotransmitter pathology, we have determined concentrations of GABA, glutamate, and the monoamine neurotransmitters in several brain regions in these mice and control animals at times before and after the emergence of the behavioural phenotype. In contrast to the findings in HD, striatal GABA was unaffected, although a deficit was observed in the cerebellum, consistent with a dysfunction of Purkinje cells. Losses of the monoamine transmitters were observed, some of which are not seen in HD. Thus, 5-hydroxytryptamine and, to a greater extent, 5-hydroxyindoleacetic acid levels were diminished in all brain regions studied, and noradrenaline was particularly affected in the hippocampus. Dopamine was decreased in the striatum in older animals, parallelling evidence for diminished dopaminergic activity in HD.

Intranuclear inclusions in subtypes of striatal neurons in Huntington's disease transgenic mice.

R6/2 transgenic mice express exon 1 of an abnormal human Huntington's disease (HD) gene and develop a neurological phenotype similar to HD. These mice develop ubiquitinated neuronal intranuclear inclusions (NII) which might play a central role in the pathophysiology of HD. We studied the distribution of NII in subpopulations of striatal neurons in 12-week-old R6/2 transgenic mice using fluorescent double label immunohistochemistry. We observed that most of the Calbindin-D28K positive projection neurons (89%) and the Parvalbumin positive interneurons (86%) showed ubiquitinated NII. In interneurons, however, which contain either choline acetyltransferase, neuronal nitric oxide synthase, or Calretinin, the frequency of NII was much lower (22%, 8%, 9%, respectively). Our data suggest that subpopulations of striatal neurons differ remarkably in their capability of forming ubiquitinated NII. Interneurons which are known to resist neurodegeneration in HD show less NII.

Transgenic mice in the study of polyglutamine repeat expansion diseases.

An increasing number of neurodegenerative diseases, including Huntington's disease (HD), have been found to be caused by a CAG/polyglutamine expansion. We have generated a mouse model of HD by the introduction of exon 1 of the human HD gene carrying highly expanded CAG repeats into the mouse germ line. These mice develop a progressive neurological phenotype. Neuronal intranuclear inclusions (NII) that are immunoreactive for huntingtin and ubiquitin have been found in the brains of symptomatic mice. In vitro analysis indicates that the inclusions are formed through self aggregation via the polyglutamine repeat into amyloid-like fibrils composed of a cross beta-sheet structure that has been termed a polar zipper. Analysis of patient material and other transgenic lines has now shown NII to be a common feature of all of these diseases. In the transgenic models, inclusions are present prior to the onset of symptoms suggesting a causal relationship. In contrast, neurodegeneration occurs after the onset of the phenotype indicating that the symptoms are caused by a neuronal dysfunction rather than a primary cell death.

Striking changes in anxiety in Huntington's disease transgenic mice.

Huntington's disease transgenic mice were tested in the elevated plus-maze test of anxiety at 6, 8, 10 and 12 weeks of age. At all ages, they showed significant and striking increases in the percentages of open arm entries and time spent on the open arms, compared with their normal littermates, indicating reduced anxiety. These increases were not secondary to a non-specific stimulant effect, since the transgenic mice made fewer closed arm entries, significantly so from 10 weeks of age. The mice were also tested in the holeboard, which provides measures of locomotor activity and directed exploration. From 8 weeks of age, the Huntington's mice were significantly less active than their normal littermates and made fewer exploratory head-dips. The increased open arm activity in the elevated plus-maze cannot therefore be secondary to increased exploration in the transgenic mice. In order to determine whether the reduced anxiety was due to differences in benzodiazepine receptor function, the mice were challenged with the benzodiazepine receptor antagonist, flumazenil. The results indicated that some of the reduced anxiety could be attributed to the presence of an endogenous anxiolytic ligand.

Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human huntington disease gene.

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9-11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-D-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not gamma-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

Striatal transplantation in a transgenic mouse model of Huntington's disease.

Striatal grafts have been proposed as a potential strategy for striatal repair in Huntington's disease, but it is unknown whether the diseased brain will compromise graft survival. A transgenic mouse line has recently been described in which hemizygotes with an expanded CAG repeat in exon 1 of the HD gene exhibit a progressive neurological phenotype similar to the motor symptoms of Huntington's disease. We have therefore evaluated the effects of the transgenic brain environment on the survival, differentiation, and function of intrastriatal striatal grafts and undertaken a preliminary analysis of the effects of the grafts on the development of neurological deficits in the host mice. Hemizygote transgenic and wild-type littermate female mice received striatal grafts at 10 weeks of age and were allowed to survive 6 weeks. Normal healthy grafts were seen to survive and differentiate within the striatum of transgenic mice in a manner comparable to that seen in control mice. The transgenic mice exhibited a progressive decline in body weight from 9 weeks of age and a progressive hypoactivity in an open field test of general locomotor behavior. Although striatal grafts exerted a statistically significant influence on several indices of this impairment, all behavioral effects were small and did not exert any clinically relevant effect on the profound neurological deficiency of the transgenic mice.