Abstract
Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour.
Highlights
Lesch-Nyhan Disease (LND) is a neurological disorder in humans that primarily affects men and is caused by mutations in the X-linked Hprt gene encoding hypoxanthine phosphoribosyltransferase (HPRT)[1,2]
In this report we describe the generation of a line of HPRT-deficient rats that exhibit metabolic and dopamine deficits associated with Lesch-Nyhan disease (LND) and should, provide a useful alternative experimental foundation for pharmacological and genetic studies into how HPRT deficiency affects the nervous system and behaviour
The Hprt targeting vector contained 2.5 kb 5′ and 2.2 kb of 3′ genomic DNA surrounding a phosphoglycerate kinase (PGK) promoter-driven neomycin selection cassette flanked by frt recombination sites, replacing exons 7 and 8 of rat Hprt (Fig. 1a)
Summary
Lesch-Nyhan Disease (LND) is a neurological disorder in humans that primarily affects men and is caused by mutations in the X-linked Hprt gene encoding hypoxanthine phosphoribosyltransferase (HPRT)[1,2]. A subset of patients carrying mutations in HPRT suffers from hyperuricaemia but does not exhibit significant neurological deficits This could imply that loss of HPRT activity affects other important molecular pathways, a notion that is supported by the finding that the basal ganglia within the brains of LND patients have markedly reduced levels of the neurotransmitter dopamine[4]. The significance of this deficit is not entirely clear, but depletion of catecholamine- (including dopamine) containing neurons in the brains of newborn rats can induce self-injurious behaviour upon administration of L-DOPA5. In this report we describe the generation of a line of HPRT-deficient rats that exhibit metabolic and dopamine deficits associated with LND and should, provide a useful alternative experimental foundation for pharmacological and genetic studies into how HPRT deficiency affects the nervous system and behaviour
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