Laura Mangiarini

Formation of Neuronal Intranuclear Inclusions Underlies the Neurological Dysfunction in Mice Transgenic for the HD Mutation

Huntingtons disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.

Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntingtons disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimers disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.

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

Striatal grafts have been proposed as a potential strategy for striatal repair in Huntingtons 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 Huntingtons 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.

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

Abstract: 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.

Amyloid-like inclusions in Huntington's disease.

Huntingtons 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 Huntingtons 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 Huntingtons 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 Huntingtons disease cases, two Alzheimers 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 Huntingtons disease brain tissue possess an amyloid-like structure, suggesting parallels with other amyloid-associated diseases such as Alzheimers and prion diseases.

Transgenic Mice in the Study of Polyglutamine Repeat Expansion Diseases

An increasing number of neurodegenerative diseases, including Huntingtons 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 β‐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

Huntingtons 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 Huntingtons 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.

Identification of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage

Abstract The Huntington’s disease mutation has been identified as a CAG/polyglutamine repeat expansion in a large gene of unknown function. In order to develop the transgenic systems necessary to uncover the molecular pathology of this disorder, it is necessary to be able to manipulate highly expanded CAG repeats in a cloned form. We have identified a patient with an expanded allele of greater than 170 repeat units and have cloned the mutant allele in the lambda zap vector. The recovery of highly expanded repeats after clone propagation was more efficient when repeats were maintained as lambda phage clones rather than as the plasmid counterparts. Manipulation of the repeats as phage clones has enabled us to generate Huntington’s disease transgenic mice that contain highly expanded (CAG)115–(CAG)150 repeats and that develop a progressive neurological phenotype.

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 Huntingtons disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntingtons 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. J. Comp. Neurol. 430:485–500, 2001.

Detection of polyglutamine aggregation in mouse models

Publisher Summary The first steps in the molecular pathogenesis of HD are beginning to be unraveled. The huntingtin protein is subjected to an initial cleavage step that releases an N-terminal fragment. The precise nature of this fragment and the cleavage mechanism are unknown, although caspase 3 has been proposed to fill this role. The N-terminal fragment can move into the nucleus, but once again, the mechanism by which this occurs is unknown. Nuclear inclusions have been described in juvenile onset HD with a frequency of 38–50% in cortical neurons, whereas dystrophic neurites (axonal inclusions) are reported to predominate in the adult form of the disease. Inclusions are observed in brain regions not traditionally associated with neuro-degeneration. Therefore, the presence of an inclusion does not necessarily lead to cell death and symptoms may be caused by dysfunction in brains regions not previously associated with HD. Dystrophic neurites were also identified in layer VI of the cortex of an individual who was presymptomatic for HD and had died of other causes. This one case suggests that, as in mice, inclusions may form prior to the onset of symptoms. Although the evidence is currently circumstantial, the early detection of inclusions suggests that they may be causative of the disease symptoms. A major application of the R6 transgenic lines is to test drugs that slow down or prevent poly(Q) aggregation. In order to demonstrate causality, it will be necessary to show that by slowing down or preventing the formation of inclusions, the onset of symptoms is correspondingly prevented or delayed. This chapter also provides protocols by which the appearance of aggregates can be monitored.