Elsevier

Brain Research

Volume 1039, Issues 1–2, 28 March 2005, Pages 14-21
Brain Research

Research report
Alterations in dopamine and benzodiazepine receptor binding precede overt neuronal pathology in mice modelling early Huntington disease pathogenesis

https://doi.org/10.1016/j.brainres.2005.01.029Get rights and content

Abstract

Huntington disease (HD) is an inherited, late onset, progressive neurodegenerative disorder. Primary degeneration appears to selectively occur in striatal medium spiny neurones but this is most likely preceded by a period of neuronal dysfunction. Altered levels of neurotransmitter receptors may disrupt neuronal function and contribute to a toxic environment within the brain. In the present study, a knock-in HD mouse modelling early stages of the disease was used to determine whether alterations in neurotransmitter receptor densities occurred before overt neuronal loss. Receptor autoradiography demonstrated reduced dopamine D2 and increased benzodiazepine receptor binding in the striatum of HD animals compared to wild-type littermates. The density of benzodiazepine receptor binding was also increased in the cerebral cortex of the HD mice. Changes in opioid and dopamine D1 receptor densities were more subtle and influenced by the genetic background of the mice. Our findings are consistent with the hypothesis that alterations in neurotransmitter receptor density precede cell loss and may be an active cellular response to the initial stages of HD pathogenesis.

Introduction

The progressive neurodegenerative disorder, Huntington disease (HD), is an autosomal dominant disease caused by the expansion of a polymorphic CAG triplet repeat sequence within exon 1 of the HD gene [17]. The expansion mutation results in an extended polyglutamine stretch within the N-terminus of the ubiquitously expressed protein called huntingtin [17]. The initial pathological basis of HD is selective degeneration of the medium spiny neurones within the striatum [16], although progression of the disease is associated with degeneration of additional brain regions, most prominently, the cerebral cortex [43]. Currently, it is not understood how the presence of mutant huntingtin leads to the cell-selective aspects of HD pathology.

The profound neuronal loss observed in end-stage disease must clearly be a major causal factor in the clinical picture. However, the disease course is so prolonged that vulnerable neurones may be disposed to long periods of dysfunction prior to cell death. The demonstration that a small number of symptomatic HD patients show no overt neuropathology (Grade 0) [42], [43] argues that massive striatal cell loss is not an absolute pre-requisite for onset of disease. A number of functional imaging studies using positron emission tomography (PET) have demonstrated reduced striatal glucose metabolism in the early stages of human HD [1], lending support to the idea that synaptic activity and, therefore, neuronal function is compromised early in HD. The precise nature of the cellular or biochemical changes that mediate neuronal dysfunction is not well understood. However, since neurotransmitters and their receptors have a central role in maintaining the normal operation of neostriatal circuitry, some effort has been invested in determining whether neurotransmitter systems are altered in HD brain tissue.

Studies of human postmortem brain tissue from HD cases have indicated changes in a variety of neurotransmitter receptors, including those associated with dopamine, acetylcholine, GABA and glutamate [9], [10], [15], [31]. Although some early stage cases have been investigated [15], it is possible that the deficits detected may reflect loss of specific sub-populations of neurones rather than a primary role in the disease process. The advent of functional imaging studies has allowed investigation of HD patients throughout their disease course. These studies have indicated that altered brain neurochemistry is present early; PET studies have revealed reduced levels of dopamine D2 receptors in the caudate and putamen of asymptomatic mutation carriers, coincident with reduced glucose metabolism [2], [3]. Studies using PET ligands have also showed altered opioid and benzodiazepine receptor binding early in HD [19], [46]. Whilst supporting the hypothesis that primary deficits in neurotransmitter systems may contribute to neuronal dysfunction in early HD, subtle cell loss could still account for the deficits.

In the past, one of the obstacles hampering studies in this field of was the paucity of appropriate tissue for analysis. However, the development of genetic models of HD provides in vivo systems that can be used to examine pathological cascades that culminate in clinical symptoms. Accordingly, we have used a knock-in HD mouse model [38] to investigate molecular and cellular changes involved in early HD pathogenesis. The insertion of a perfect CAG repeat tract into exon 1 of the mouse Hdh gene has provided the opportunity to explore the consequences of the HD mutation in its appropriate genomic and protein context. Previous studies of these mice have demonstrated several phenotypic changes including behavioural and motor abnormalities [21], [38], as well as abnormalities of long-term potentiation in the hippocampus [41]. The phenotypic and cellular changes appear to occur before frank neurodegeneration as histological and immunohistochemical analyses have failed to reveal any evidence of overt neuronal loss in the striatum of 18-month-old HD mice [38].

In the present study, we have determined the density of dopamine, opioid and benzodiazepine receptor binding sites in the brains of knock-in HD mice using quantitative ligand binding autoradiography. The ligands selected for this study were based on those used previously in human brain imaging studies of pre-end stage disease patients and we focused on the striatum as the region of primary pathology. In order to investigate the presence of potential genetic modifiers of the early disease process, a post hoc subgroup analysis of animals with different genetic backgrounds was performed.

Section snippets

Animals

The generation of the knock-in HD mice used in the current study has been described previously [38]. All experiments were performed on female progeny of the founder mouse line Hdh4/Q80 inbred onto either a C57BL/6 (N6–7 generation) or FVB/N (N4 generation) genetic background. A total of 15 HD and 15 wild-type mice was used. The genotype of the mice was determined by PCR analysis of tail DNA biopsies using standard procedures [38]. All experiments were carried out using 17- to 18-month-old

Results

Autoradiograms of total binding for the different ligands are presented in Fig. 1 for illustrative purposes. On visual inspection, differences in the levels of binding between HD and wild-type mice were not marked. However, the quantitative analysis data revealed there to be statistically significant differences between the two groups.

Discussion

The present study demonstrates that 18-month-old knock-in HD mice expressing full-length mutant huntingtin appropriately have reduced levels of D2 receptor binding sites in the striatum and increased levels of benzodiazepine receptor binding sites in the striatum and cerebral cortex. There is also a trend towards increased opioid binding densities in the striatum and, to a lesser extent, in the cortex of the HD mice, although these changes do not reach statistical significance. All these

Acknowledgments

The authors are grateful to Colin Hughes, Dennis Duggan, Margaret Ennis and staff at the Wellcome Surgical Institute for technical assistance and The University of Glasgow Dynamic Mutation Group and Professor J. McCulloch for helpful discussion. This work was funded by the Hereditary Disease Foundation. L.K. was supported by a studentship from The Huntington's Disease Association of Great Britain.

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