Identification of elevated urea as a severe, ubiquitous metabolic defect in the brain of patients with Huntington's disease

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Highlights

  • GC-MS was used to measure urea in 11 human brain regions of Huntington's disease patients.

  • Urea is significantly elevated in all region studied in the Huntington's disease brain.

  • Urea levels are sufficient to generate cytotoxicity in the brain.

Abstract

Huntington's disease (HD) is a neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein through the lengthening of a polyglutamine tract and initiates a cascade that ultimately leads to dementia and premature death. However, neurodegeneration typically manifests in HD only in middle age, and processes linking the causative mutation to brain disease are poorly understood. Here, our objective was to elucidate further the processes that cause neurodegeneration in HD, by measuring levels of metabolites in brain regions known to undergo varying degrees of damage. We applied gas-chromatography/mass spectrometry-based metabolomics in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine controls. Unexpectedly, a single major abnormality was evident in all eleven brain regions studied across the forebrain, midbrain and hindbrain, namely marked elevation of urea, a metabolite formed in the urea cycle by arginase-mediated cleavage of arginine. Urea cycle activity localizes primarily in the liver, where it functions to incorporate protein-derived amine-nitrogen into urea for recycling or urinary excretion. It also occurs in other cell-types, but systemic over-production of urea is not known in HD. These findings are consistent with impaired local urea regulation in brain, by up-regulation of synthesis and/or defective clearance. We hypothesize that defective brain urea metabolism could play a substantive role in the pathogenesis of neurodegeneration, perhaps via defects in osmoregulation or nitrogen metabolism. Brain urea metabolism is therefore a target for generating novel monitoring/imaging strategies and/or therapeutic interventions aimed at ameliorating the impact of HD in patients.

Introduction

Huntington's disease is an autosomal dominant genetic disorder caused by an expanded CAG repeat in exon 1 of the HTT gene, which is located on chromosome 4 and encodes the huntingtin protein [1]. Biochemical pathways in which alterations have been linked to neurodegeneration in HD include cell-stress responses, apoptosis, several metabolic pathways including the ubiquitin-proteasome system and protein trafficking/endocytosis [2].

In manifest HD, progressive degeneration of the brain leads ultimately to symptoms including involuntary body movements (chorea), disturbances of speech, and progressive dementia [3]. Although the caudate-putamen and cerebral cortex are known primary targets of neurodegeneration in HD, it has been reported that several other brain regions are also affected in this complex disease process [4].

Besides the well-characterised neuropathology, several lines of evidence point to a widespread metabolic perturbation in HD. There is evidence that this mechanism is not limited to the basal ganglia and associated neural circuitry; rather, it is thought to be pervasive, affecting many brain regions in HD-gene carriers [5], [6]. Furthermore, the existence of a systemic metabolic defect is also supported in HD [7], [8], [9].

In order to study the effects of HD on metabolism across the affected brain, we chose to apply gas chromatography/mass spectrometry-based metabolomics to detect and characterise putative alterations in metabolite levels in short post-mortem-delay brain tissue of patients and matched controls, using a case-control approach.

Eleven regions of the human brain were analysed here. The putamen and motor cortex were selected as they are most severely affected in advanced disease whereas sensory cortex, globus pallidus, cingulate gyrus and substantia nigra represent regions moderately affected in the HD brain [10], [11]. Finally, hippocampus, entorhinal cortex, cerebellum, middle frontal gyrus and middle temporal gyrus were included as they are not usually associated with overt degeneration.

Section snippets

Acquisition of human brains

Whole brains from nine HD patients and nine matched controls were obtained from the New Zealand Neurological Foundation Human Brain Bank, in the Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand. All procedures in this study were approved by the University of Auckland Human Participants Ethics Committee with informed consent from all families. The quality of HD brain tissue acquired by the Auckland brain bank is uniformly high as

Results and discussion

We measured urea as mmol/kg of wet brain-tissue weight, in 11 brain regions from 9 patients with HD and 9 matched controls. Cases and controls were matched for male:female ratio, age, and post-mortem delay (Table 1). Brain-weights were significantly lighter in cases than in controls (P = 0.01). HD cases were all heterozygous for HTT mutations, and elevated CAG repeat-lengths ranged between 40 and 47 (Table 2).

Reproducibility of the GC-MS-derived measurements was verified by monitoring urea

Conflict of interest

None.

Acknowledgements

We thank all the families of patients with Huntington's disease in New Zealand who so generously supported this research through the donation of brain tissue to the NZ Neurological Foundation Human Brain Bank in the Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, New Zealand. This work was supported by the CHDI Foundation (A-8247); the Endocore Research Trust (61047); the Maurice and Phyllis Paykel Trust (3627036); the Neurological Foundation of New

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