Department of Medical and Molecular Genetics, Indiana University School of Medicine, IB 130, 975 West Walnut Street, Indianapolis, IN 46202-5251, USA, ¹Purine Research Laboratory, Guy's Hospital, London SE1 9RT, UK and ²Departments of Cell Biology, Neurobiology and Anatomy, and Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA

Received May 3, 1996; Revised and Accepted July 8, 1996

Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in humans results in the Lesch-Nyhan syndrome which is characterized, among other features, by compulsive self-injurious behavior. HPRT-deficient mice generated using mouse embryonic stem cells exhibit none of the behavioral symptoms associated with the Lesch-Nyhan syndrome. Administration of drugs that inhibit adenine phosphoribosyltransferase (APRT) in HPRT-deficient mice has produced the suggestion that deficiency of APRT in combination with HPRT-deficiency in mice may lead to self-mutilation behavior [C. L. Wu and D. W. Melton (1993) Nature Genet. 3, 235-240]. To test this proposition, we bred HPRT-APRT-deficient mice. Although the doubly-deficient mice excrete adenine and its highly insoluble derivative, 2,8-dihydroxyadenine, which are also associated with human APRT deficiency, additional abnormalities or any self-injurious behavior were not detected. Thus, APRT-HPRT-deficient mice, which are devoid of any purine salvage pathways, show no novel phenotype and are not a model for the behavioral abnormalities associated with the Lesch-Nyhan syndrome as previously suggested.

INTRODUCTION

The Lesch-Nyhan syndrome is an X-linked disorder characterized by hyperuricemia, choreoathetosis, spasticity, mental retardation and, most strikingly, compulsive self-injurious behavior (1 ). It results from a complete lack of hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8), the enzyme that converts hypoxanthine or guanine to inosine monophosphate or guanosine monophosphate, respectively. The role of HPRT deficiency in generating the behavioral abnormalities seen in Lesch-Nyhan syndrome is not understood.

Several attempts to generate animal models for Lesch-Nyhan syndrome using pharmacological or surgical interventions have done little to link purine metabolism to behavioral abnormalities. In addition, HPRT-deficient mouse embryonic stem (ES) cells have been used to produce HPRT-deficient mice (2 ,3 ). Although these mice were mutant for the same gene that is mutant in humans with Lesch-Nyhan syndrome, they were essentially normal and healthy with only subtle changes in brain dopamine levels (4 ,5 ).

Since differences in purine metabolism between rodents and man could be responsible for the different consequences of HPRT deficiency, Wu and Melton investigated the effect of reduced adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) activity on HPRT-deficient mice (6 ). APRT, a purine salvage enzyme similar to HPRT, converts adenine to adenosine monophosphate (7 ). Wu and Melton treated five 9-12 month-old HPRT-deficient mice with the APRT enzyme inhibitor 9-ethyladenine. All five of the treated mice, but none of the control mice, were reported to develop self-inflicted injuries caused by overgrooming, within 48-130 days after treatment began. Self-injurious behavior was defined to include all grooming with fore and hind legs, nibbling and biting. A second set of younger (6-8 week-old) HPRT-deficient mice injected with 9-ethyladenine or caffeine were also reported to show an increase in self-injurious behavior during the treatment period. They concluded from their drug studies that APRT does play a more important role in purine metabolism in mouse as compared with man and that partial inhibition of APRT activity produces a model for human Lesch-Nyhan syndrome. At the time that this study was performed, APRT-deficient mice were not available to confirm the role of this enzyme in preventing the behavioral symptoms associated with total HPRT deficiency.

Recently, we have generated APRT-deficient mice using homologous recombination in ES cells and an appropriate breeding strategy (8 ). APRT-deficient mice excrete adenine and its insoluble oxidation product, 2,8-dihydroxyadenine (DHA) which is characteristic of the human deficiency. In addition, these mice develop the DHA kidney stones and renal failure seen in untreated APRT-deficient humans. APRT-deficient mice are, therefore, an excellent model for the clinical symptoms of the deficiency.

To test the hypothesis that APRT deficiency, in conjunction with HPRT deficiency in mice, can produce the behavioral symptoms of Lesch-Nyhan syndrome, we bred the nonfunctional APRT allele into an HPRT-deficient mouse background. Mice deficient in both APRT and HPRT activity excreted adenine and DHA but did not exhibit any anatomical defects or spontaneous behavioral abnormalities; therefore, the hypothesis that mice are more dependent than humans on APRT for purine salvage is not supported and these mice are not a model for the behavioral abnormalities associated with Lesch-Nyhan syndrome as previously suggested.

RESULTS

APRT-deficient mice were produced as previously described (8 ). HPRT-deficient mice were produced by blastocyst injection using Hprt^-/0 E14TG2a ES cells (8 ) and the appropriate breeding of chimeric mice and their offspring. The Aprt null allele was introduced into the HPRT-deficient background as described in Materials and Methods. Southern hybridization analysis or polymerase chain reaction (PCR) amplification of genomic DNA from tail biopsies from 3-6 week-old mice were used to determine the Aprt genotype. Culture of tail biopsies in hypoxanthine-aminopterin-thymidine (HAT) or 6-thioguanine (TG) containing medium was used to confirm the HPRT-deficient phenotype.

Hprt^-/-(-/0) Aprt^+/- mice were healthy and fertile. Crosses between Hprt^-/-(-/0) Aprt^+/- mice produced an average of seven pups per litter. One hundred and fourteen offspring (54% male and 46% female) from these matings were analyzed for their Aprt genotype. HPRT phenotype was determined by tail cell culture in selective medium only in randomly selected mice, since all offspring were expected to be HPRT-deficient. Twenty-five percent were Hprt^-/-(-/0) Aprt^+/+, 55% were Hprt^-/-(-/0) Aprt^+/- and 20% were Hprt^-/-(-/0) Aprt^-/-. These frequencies do not significantly differ from the expected. This suggests that the combined deficiency of HPRT and APRT activity has no effect on the viability of the mice either prenatally or prior to weaning.

Hprt^-/-(-/0) Aprt^-/- mice do not exhibit any anatomical abnormalities or obvious behavioral changes and are indistinguishable from their littermates. Both male and female doubly-deficient mice are fertile and demonstrate normal reproductive capabilities. None of the more than 23 doubly-deficient mice became ill or died during the first 8 months of observation.

HPRT and APRT activities were assayed in red blood cell (RBC) lysates from Hprt^-/-(-/0) Aprt^-/- mice and their littermates. As seen in Table 1 , Aprt wild-type and heterozygous mice exhibited significant APRT activity, while mice homozygous for the Aprt null allele exhibited enzyme activity that was indistinguishable from background. None of the mice exhibited measurable HPRT activity, as expected from the Hprt^-/-(-/0) Aprt^+/- cross. HPRT activity was measured in Hprt normal mice as a control. Lack of APRT activity in liver extracts confirmed the data from RBC lysates.

Urine samples from 6 week-old mice were analyzed for purine metabolites by high pressure liquid chromatography (Table 2 ). The total amount of adenine metabolites in the urine (adenine and DHA) of doubly-deficient mice was approximately one half of that found in mice solely deficient in APRT activity (0.42 +- 0.16 versus 0.82 +- 0.32 mmol/mmol creatinine) (8 ). The ratio of adenine to DHA was 1:1.6, which is similar to the 1:1.5 ratio typically seen in APRT-deficient humans (7 ) but significantly lower than the 1:3 ratio seen in mice solely deficient in APRT activity (8 ). There were no significant differences in the levels of urinary hypoxanthine, xanthine and uric acid between doubly-deficient mice and their littermates. This is in contrast with mice solely deficient in APRT activity which excrete approximately one half the amount of uric acid excreted by their APRT wild-type and heterozygous littermates (8 ).

Table 1 APRT and HPRT activity of wild-type, heterozygous and APRT-deficient mice bred into an HPRT-deficient background

Genotype	RBC lysates		Liver extracts
	APRT activity	HPRT activity	APRT activity
Hprt+/+(+/0) Aprt+/+	2.87 +- 1.58 (10)	0.63 +- 0.21 (7)	5.81 +- 4.66 (9)
Hprt-/-(-/0) Aprt+/+	3.59 +- 2.13 (8)	0.01 +- 0.02 (7)	10.97 +- 2.99 (8)
Hprt-/-(-/0) Aprt+/-	1.40 +- 0.56 (11)	0.02 +- 0.02 (10)	4.69 +- 0.95 (8)
Hprt-/-(-/0) Aprt-/-	0.01 +- 0.01 (10)	0.01 +- 0.02 (10)	0.08 +- 0.13 (6)

The enzyme activities are reported in nmoles of substrate (either adenine or hypoxanthine) converted/h/mg protein. Numbers represent the mean +- the standard deviation. The numbers in parentheses indicate the number of mice assayed. Data from male and female mice are combined.

Table 2 Metabolites in the urine of 6 week-old wild-type, heterozygous and APRT-deficient mice bred into an HPRT-deficient background

Genotype	Adenine	DHA	Uric acid	Hypoxanthine	Xanthine
Hprt+/+(+/0) Aprt+/+ (6)a	<0.01	<0.01	0.49 +- 0.16	<0.01	<0.01
Hprt+/+(+/0) Aprt-/- (4)a	0.20 +- 0.07	0.62 +- 0.32	0.16 +- 0.14	<0.01	<0.01
Hprt-/-(-/0) Aprt+/+ (6)	<0.01	<0.01	0.41 +- 0.07	0.01 +- <0.01	0.01 +- <0.01
Hprt-/-(-/0) Aprt+/- (5)	<0.01	<0.01	0.50 +- 0.15	0.01 +- <0.01	0.01 +- 0.01
Hprt-/-(-/0) Aprt-/- (8)	0.16 +- 0.12	0.26 +- 0.10	0.30 +- 0.10	0.03 +- 0.03	0.03 +- 0.02

The concentration of metabolites is reported in mmoles product per mmole creatinine. The numbers represent the mean +- the standard deviation. The numbers in parentheses indicate the number of mice assayed. Data from male and female mice are combined.^aData obtained from reference 8.

Hprt^-/-(-/0) Aprt^-/- mice were examined histopathologically at 6 and 12 weeks of age. Formalin-fixed, paraffin-embedded tissues from all major organ systems were sectioned and stained with hematoxylin and eosin. All doubly-deficient mice of both age groups (three 6 week-old and three 12 week-old mice) showed mild nephritis associated with DHA crystal formation and some dilation or inflammation of the renal pelvis (data not shown). The lesions were characterized by small DHA crystals in the cortex with minimal lymphocytic infiltration. In general, doubly-deficient mice appeared to have kidney damage similar to, but less severe than, mice of comparable age solely deficient in APRT activity. No significant lesions were detected in any other tissues from the doubly-deficient mice or from Hprt^-/-(-/0) Aprt^+/+ mice.

Self-injurious behavior was not observed in the Hprt^-/-(-/0) Aprt^-/- mice. In the more than 8 months that the mice have been observed, no injuries, self-inflicted or otherwise, have been detected. In addition, fighting and barbering among mice have not been observed. Since Wu and Melton (6 ) suggested that the injuries they observed resulted from excessive stereotypic behaviors, the frequency of stereotypic behaviors was determined as described in Materials and Methods. The behavior study, although not extensive, was chosen to reflect the methodology used by Wu and Melton (6 ) so that a direct comparison of mouse behaviors could be made. We observed no significant difference in the frequency of stereotypic behaviors among wild-type, APRT-deficient, HPRT-deficient and doubly-deficient mice (Table 3 ).

Table 3 Frequency of stereotypic behaviors in mice of different Hprt and Aprt genotypes

Genotype	Number of stereotypic behaviors
Hprt+/+(+/0) Aprt+/+ (11)	22.00 +- 2.93
Hprt+/+(+/0) Aprt-/- ( 6)	22.33 +- 6.28
Hprt-/-(-/0) Aprt+/+ ( 8)	24.50 +- 5.01
Hprt-/-(-/0) Aprt-/- ( 8)	24.38 +- 7.82

The numbers represent the mean +- the standard deviation. The numbers in parentheses indicate the number of mice tested. Data from male and female mice are combined. ANOVA F(3,32) = 0.49, P = 0.7.

DISCUSSION

The lack of self-inflicted wounds, elevated levels of stereotypic behaviors or spontaneous behavioral abnormalities suggests that Hprt^-/-(-/0) Aprt^-/- mice are not a model for the behaviors associated with Lesch-Nyhan syndrome. The discrepancy between our results and those of Wu and Melton (6 ) may result from the APRT inhibitors used. Caffeine and 9-ethyladenine are likely to have affected pathways unrelated to adenine salvage. Caffeine is a stimulant which affects many biological processes (9 ) and induces self-injurious behaviors in rodents at high doses (10 ). Wu and Melton's own data suggest that 9-ethyladenine elevates stereotypic behaviors in HPRT positive mice (0.32 +- 0.18 self-injurious behaviors/min) as well as HPRT-deficient mice (1.21 +- 0.42 self-injurious behaviors/min) when compared with mice treated with saline (0.11 +- 0.14 self-injurious behaviors/min) (6 ). Although the mechanism by which caffeine and 9-ethyladenine may cause the behaviors observed by Wu and Melton is of interest, it is irrelevant to the discussion of how the lack of purine salvage causes Lesch-Nyhan syndrome.

Previous neurochemical studies of HPRT-deficient mice have shown a reduction in the levels of the neurotransmitter dopamine and the activity of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, in the caudate and putamen regions of the brain, although neurotransmitter and enzyme levels in other regions of the brain were within normal limits (4 ,5 ). Reductions in the same neurotransmitter and enzyme in the caudate and putamen of human patients with Lesch-Nyhan syndrome have also been demonstrated (11 ). Recent ligand binding studies have shown that the levels of dopamine transporters are also reduced (12 ). These findings, in addition to evidence from a drug-induced, dopamine-deficient rat model of the behaviors characteristic of Lesch-Nyhan syndrome (13 ), suggest that the HPRT deficiency is associated with a relatively specific defect in the dopamine system in the basal ganglia. This is despite the fact that HPRT-deficient mice do not show any explicit behavior problems associated with a defect in this system. Over an 8 month period, we have not observed any behavioral abnormalities in the HPRT-APRT-deficient mice, but neurotransmitter studies in these mice have not yet been completed. It is possible that a complete battery of neurochemical and neurobehavioral testing will elucidate subtle defects in these mice.

Additionally, our results suggest that APRT does not play a role in mice that is different from its role in man. Although the doubly-deficient mice will be closely monitored to determine if additional symptoms develop with advanced age, it is likely that the only significant characteristic associated with the double deficiency is DHA lithiasis. This is seen, at a somewhat greater extent, in mice that are solely deficient in APRT.

The less aggressive nature of the kidney disease in the doubly-deficient mice suggests that HPRT deficiency may be mitigating some of the effects of APRT deficiency. In wild-type mice (and humans), the majority of hypoxanthine is returned to the nucleotide pool by the activity of HPRT. Unsalvaged hypoxanthine is converted by xanthine dehydrogenase (XDH, EC 1.2.3.2) to xanthine and, subsequently, uric acid. There is a marked increase in the excretion of hypoxanthine and uric acid in human HPRT deficiency (1 ), although we observed only a minimal increase in urinary hypoxanthine in murine HPRT deficiency. In the absence of APRT, excess adenine is oxidized by XDH to DHA even though hypoxanthine is ~1000-fold better substrate for XDH. It is likely that even small increases in hypoxanthine resulting from HPRT deficiency effectively compete with adenine as a substrate for XDH. Thus, the lower levels of urinary adenine and DHA, the reduced renal damage and the approximately wild-type levels of urinary uric acid in HPRT-APRT-deficient mice may be due to the increased availability of hypoxanthine. Further analysis of Hprt^-/-(-/0) Aprt^-/- mice may provide unique insight into the regulation of purine metabolism in the absence of purine salvage.

MATERIALS AND METHODS

Breeding and maintenance of mice

To derive mice deficient for both HPRT and APRT activity, the following breeding scheme was utilized. Female Hprt^-/- Aprt^+/+ mice were mated with male Hprt^+/0 Aprt^+/- mice which had been bred into a C57Bl/6 background. Hprt^-/0 Aprt^+/- male mice were mated to their Hprt^-/- Aprt^+/+ mothers. Hprt^-/-(-/0) Aprt^+/- offspring from this mating were crossed to generate doubly-deficient (Hprt^-/-(-/0) Aprt^-/-) mice. All mice were maintained in a pathogen-free barrier facility at Indiana University School of Medicine.

Southern hybridization analysis and PCR analysis

Southern hybridization analysis was performed as previously described (8 ,14 ). PCR amplification of genomic DNA was done in a total reaction volume of 100 [mu]l containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% w/v gelatin, 0.2 mM each deoxyribonucleotide and 300 ng each of primers MA009 (5'-TCCCACAACCTTCCCTCCTTAC-3'), NEO-4 (5'-TGCCTGCTTGCCGAATATCATGGT-3') and MA010 (5'-CACCAAGCAGTTCCTAGTGCT-3'). The reactions were run for 35 cycles of 94^oC for 60 s and 63^oC for 120 s.

Mouse tail cell cultures

Approximately 1 cm of mouse tail was minced and incubated with gentle rotation for 45 min at 37^oC in 0.2 ml of Dispase neutral protease-collagenase D (4 mg each in 100 ml medium, Boehringer Mannheim). Dulbecco's Modified Eagle's Medium (high glucose, Life Technologies, Inc.) (1 ml) supplemented with 15% fetal bovine serum, 2 mM L-glutamine, 1* MEM nonessential amino acids and penicillin-streptomycin (5000 U each/ml) was added and the cells dissociated by gentle pipetting before overnight incubation in a 24-well dish at 37^oC, 5% CO₂. The next day, the unattached cells were centrifuged for 15 min at 1000 r.p.m., resuspended in 1 ml of fresh culture medium, and replated. When cells became confluent, they were passaged into dishes containing culture medium supplemented with either 6-TG (30 [mu]M) or HAT (hypoxanthine, 100 [mu]M; aminopterin, 0.4 [mu]M; thymidine, 16 [mu]M).

APRT and HPRT assays

APRT assays were performed as described (8 ). HPRT assays were performed in similar manner except that 8-¹⁴C-labeled hypoxanthine ([approx]50 mCi/mmol, Amersham Life Sciences) replaced adenine as the substrate.

Analysis of purine metabolites in urine

High pressure liquid chromatography analyses were performed as described (8 ,15 ).

Pathology and histopathology

Histological examination was performed on representative sections from eye, harderian gland, salivary gland, mandibular muscle, cervical lymph node, heart, lung, thymus, diaphragm, adrenal gland, kidney, urinary bladder, ureter, liver, gall bladder, spleen, stomach, duodenum, pancreas, jejunum, ileum, colon, skin, quadriceps muscle, cerebrum and cerebellum. Tissues were fixed in 10% neutral buffered formalin, dehydrated through a gradient of alcohols, and embedded in paraffin prior to staining with hematoxylin and eoxin.

Behavioral analysis of mice

Mice were routinely caged in groups of three to five and maintained on a 12 h light/dark cycle. Observation of stereotypical behaviors was performed using a 2 day testing paradigm. On day 1, mice were placed individually into clean, standard housing cages for 20 min. Mice were returned to their normal cages overnight. On day 2, mice were placed in the same cage as on day 1 and observed for 1 min every 5 min, thus allowing four observation periods per mouse during the 20 min testing session. Stereotypical behaviors counted included rearing, licking, gnawing, sway, body grooming, face washing, nose pokes and pattern locomotion. The genotypes of the 8-12 week-old mice were unknown to the trained observer to reduce observational bias.

ACKNOWLEDGEMENTS

We thank Thomas Doetschman for E14TG2a cells and the personnel of the Division of Comparative Pathology, University of Cincinnati, for their help in preparing samples for histological examination. This work was supported by NIH grant DK38185.

REFERENCES

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15 Simmonds, H.A., Duley, J.A. and Davies, P.M. (1991) In Hommes, F.A. (ed.) Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual. Wiley-Liss, London, pp. 397-425.

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