Production and characterization of murine models of classic and intermediate maple syrup urine disease
© Homanics et al; licensee BioMed Central Ltd. 2006
Received: 15 February 2006
Accepted: 31 March 2006
Published: 31 March 2006
Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model.
To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels.
By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model.
These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease.
Maple Syrup Urine Disease (MSUD) is a genetic disorder caused by a deficiency of branched-chain keto acid dehydrogenase (BCKDH), a mitochondrial multienzyme complex responsible for the oxidative decarboxylation of branched-chain keto acids derived from branched-chain amino acids (BCAA), leucine, isoleucine and valine (for review, see: ).
Patients with MSUD, depending on the mutation, show variable degrees of enzyme deficiency leading to several different clinical phenotypes . Approximately 75% of MSUD patients have the classic form of the disease with BCKDH activity in the range of 0–2% of normal . These patients show markedly elevated levels of BCAA in blood and other body fluids . Besides the classic form, there are other variants of the disease. Patients with the intermediate form of the disease show BCKDH activity in the range of 3–30% of normal. In such patients the onset of the disease is delayed, but there are persistently elevated levels of BCAA . Patients with the intermittent form of MSUD show BCKDH activity in the range of 5–20% and during the asymptomatic phase the blood BCAA levels are normal . The overall incidence of MSUD in the general population is 1:185,000 , and in certain population groups, such as Mennonites of Pennsylvania, the incidence is estimated to be as high as 1:176 .
The BCKDH complex, the deficient enzyme in MSUD, consists of three catalytic proteins, a decarboxylase (E1), a dihydrolipoyl transacylase (E2), and a dihydrolipoyl dehydrogenase (E3). The E1 component is a heterotetramer composed of two α and two β subunits . The E1 and E2 components are specific to BCKDH, whereas E3 is also used by pyruvate and α-ketoglutarate dehydrogenase complexes  and the glycine cleavage system . BCKDH is also associated with two regulatory proteins, a specific kinase and a phosphatase which regulate the activity of this enzyme through a phosphorylation (inactivation) and dephosphorylation (activation) cycle of the E1 α subunit [5, 6]. Mutations in the genes of the E1 and E2 subunits of BCKDH have been described, however, the majority of MSUD mutations identified thus far are in the E2 subunit [1, 7]. To date, cases of MSUD have not been associated with defects in the regulatory kinase and phosphatase .
Current management of MSUD patients relies on a strict lifelong dietary restriction of protein or BCAA [1, 8]. Such a dietary management of the disease, however, is not entirely satisfactory especially in times of metabolic decompensation due to infection, injuries and other stressors. In spite of dietary intervention, there is significant mortality associated with MSUD and there is a high incidence of mental retardation in survivors .
Because of the central role of the liver in amino acid metabolism and moderate/high levels of BCKDH activity in human liver [10–12], a few cases of MSUD have recently been treated by liver transplantation [13–16]. While the short-term outcome of liver transplantation is encouraging, long-term effects of this approach are not known. However, these patients are now required to take immunosuppressant drugs for the rest of their lives, often with undesirable side effects. Moreover, the cost associated with liver transplantation and the availability of donor livers are additional limiting factors for the practicality of treatment of this disease.
Because of the current unsatisfactory options for the treatment of MSUD, there is a need for improved therapies to combat this disease. An obstacle to developing novel treatments for MSUD has been the lack of a suitable animal model to perform necessary preclinical studies. Although a Hereford calf model with MSUD has been described [17–19], this model is neither readily available nor practical to perform preclinical studies. Furthermore, comparison of this animal model with human MSUD has shown some differences in the pathology of the disease , making this animal a less desirable model for the human disease.
Recently, a N-ethyl-N-nitrosourea (ENU)-induced mutant mouse that phenotypically resembles human MSUD has been described . However, the mutation in this model disrupts a splice site in the mitochondrial branched-chain aminotransferase (BCAT) gene, not in BCKDH, the deficient enzyme in MSUD. Because the mutation is not in BCKDH, the validity of this mutant mouse line for modeling human MSUD is questionable.
The objective of the present study was to create genetically engineered murine models of MSUD that mimic the pathology of the classic and intermediate variant forms of the human disease. The classic model was created by targeted inactivation of the E2 subunit of BCKDH by homologous recombination in embryonic stem (ES) cells. The model of intermediate MSUD was created by partial transgenic rescue of the E2 gene knockout. This report describes the generation and characterization of these murine models of MSUD. These models will allow for the development of novel treatment approaches, such as gene or stem cell therapies, to ultimately cure MSUD.
All studies involving animals were reviewed and approved by the University of Pittsburgh's Institutional Animal Care and Use Committee.
Gene knockout production
Production of transgenic mice
Standard molecular techniques were used to assemble the transgenic construct, pTRE-E2. This transgene contains the tetracycline responsive hCMV*-1 promoter [consisting of the tetracycline responsive element (TRE) and a minimal hCMV promoter]  from pTRE2 (Clontech Inc., Mt. View, CA), a chimeric intron from pCI (Promega) to increase message stability and expression [26, 27], a Kozak consensus sequence at the initiation codon to optimize translation , the human E2 cDNA which has been modified to contain a 4× alanine linker followed by a c-myc epitope tag at the carboxy terminus to facilitate detection, and an SV40 late polyadenylation sequence from pCI for enhanced mRNA stability and translation.
The 2.48 kb TRE-E2 transgene was purified from vector sequences following digestion with XhoI and BamHI and injected into C57BL/6J or C57BL/6J × Strain 129SvEv mouse embryos at the transgenic core facilities of the University of Pittsburgh and the University of Cincinnati, respectively. Genomic DNA from the tail of mice was screened by Southern blot analysis following digestion with EcoRI and hybridization to an ~400 bp probe derived from the SV40 portion of the TRE-E2 transgene.
Production of intermediate MSUD murine model
The various TRE-E2 transgenic lines produced were mated independently to mice that were heterozygous for both the E2 knockout and the LAP-tTA transgene [Tg(tTALap)Bjd/J; Stock 3272; The Jackson Laboratory, Bar Harbor, ME; NMRI × FVB × C57BL/6J background]. Interbreeding of animals that were heterozygous for both transgenes and the knockout resulted in the production of mice with a variety of genotypes including some animals that were homozygous for the knockout and were positive for both transgenes (we refer to this genotype as the "rescue" genotype). If our strategy for rescuing the neonatal lethal phenotype of the knockout were successful, then those homozygous knockout animals that had both transgenes would survive beyond the neonatal period.
All mice were genotyped by Southern blot analysis. Genomic DNAs prepared from tail snips were digested with an appropriate restriction enzyme, size fractionated by agarose gel electrophoresis, blotted to nylon, and probed using standard procedures.
Primary mouse embryonic fibroblasts were prepared from embryonic day ~16.5–18.5 fetuses as described . Fibroblasts were passed onto glass, fixed in 2% paraformaldehyde in PBS for 10 minutes, permeabilized in 2% paraformaldehyde containing 0.1% Triton X100 for 10 minutes and washed three times in PBS containing 0.5% BSA and 0.15% glycine, pH 7.4 (Buffer A). Following a 30 min incubation with purified goat IgG (50 (μg/ml) at 25°C and three additional washes with Buffer A, cells were incubated for 60 min with E2-specific antiserum  at 1 μg/ml followed by three washes in Buffer A and 60 minute incubation in fluorescently labeled second antibody (1–2 μg/ml). The cells were then washed six times (5 min/wash) in Buffer A and then mounted in gelvatol (Monsanto, St Louis). When livers from newborn pups were examined, fixation was by immersion in 2% paraformaldehyde followed by cryoprotection and shock freezing in liquid nitrogen cooled isopentane and sectioning (5 microns). Otherwise processing was as for the cells above (without the fixation and permabilization steps).
Amino acid analysis
Blood was collected from the retroorbital sinus or tail vein of mice and spotted on a filter paper routinely used for blood amino acid analysis for prenatal screening. Concentrations of BCAA and other amino acids in blood were determined by tandem mass spectrometry (Pediatrix Screening, Bridgeville, PA).
Assay of BCKDH activity
Livers were removed, frozen in liquid nitrogen, and stored at -80°C. At the time of enzyme assay, livers were thawed, and homogenized (1:9, w/v) in 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4. Liver homogenates were centrifuged at 600 × g for 10 min at 4°C and the supernatant fraction was saved to determine the BCKDH activity. The use of tissue homogenates was necessitated by the limited availability of liver tissue, particularly from newborn pups. BCKDH activity was determined by measuring the release of 14CO2 from α-keto [1-14C] isocaproate as described previously . The complete reaction mixture contained (final volume 1 ml) 30 mM potassium phosphate buffer, pH 7.4, 0.20 mM α-ketoisocaproate, 0.40 mM CoASH, 0.40 mM thiamin pyrophosphate, 2 mM NAD+, 2 mM dithiothreitol, 5 mM Mg2+, approximately 250,000 DPM of α-keto [1-14C] isocaproate, and 0.10 ml of liver homogenate (2–3 mg protein). Assays were carried out for 15 min at 37°C, 14CO2 was trapped in hydroxide of Hyamine, and radioactivity was determined by liquid scintillation spectrometry.
Western blot analysis
Protein extracts were isolated from homogenized liver (freshly harvested and flash frozen) of wildtype, Line 525A, and Line A transgenic mice. Protein (25 μg per sample) was analyzed by electrophoresis on a 10% SDS-PAGE Ready Gel (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (Sequi-Blot; Bio-Rad) via electroblotting. All blots were probed for E2 protein using polyclonal rabbit E2 antisera (1:5,000), which detects both mouse (~47 kD) and human (~54 kD) E2 subunits (; gift from Dr. Susan Hutson, Wake Forest University). Blots were re-probed with a rabbit anti-c-myc tag antibody (1:10,000; abCam, Cambridge, MA; cat.# ab9106-100). Blots were also re-probed with an antibody for β-actin (43 kD; 1:10,000; abCam; cat.# ab8227-50) to allow for loading comparisons. A goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000; Novus, Littleton, CO; cat.# NB730-H) was used for detection using the Western Lightning chemiluminescence reagent (Perkin Elmer, Boston, MA) and exposed to X-ray film.
All BCAA and BCKDH enzyme activity data are presented as the mean +/- the standard error of the mean (S.E.M.). Differences between genotypes were compared by Student's t test.
Production of E2 gene knockout mice
To create E2 knockout mice, we used gene targeting in mouse ES cells to disrupt the E2 gene. The overall strategy for disrupting the E2 gene is illustrated in Fig. 1A. The gene targeting construct was designed to replace a 1.67 kb EcoRV/Smal genomic DNA fragment encompassing part of Exon 4 and all of Exon 5 with the PGKneo selectable marker cassette. Of 522 ES cell clones screened for targeting by Southern blot analysis, 29 (5%) displayed the predicted restriction fragment length polymorphisms indicative of gene targeting at the E2 locus. As illustrated in Fig. 1A and 1B, an E2 Exon 6 specific probe, which is external to the targeting construct, hybridizes to only a ~16 kb BglI restriction fragment from the wild type allele in the parental R1 ES cell line. In correctly targeted ES cells, this probe also hybridizes to a ~11 kb BglI restriction fragment. Targeting was confirmed with several additional restriction enzymes and probes (data not shown).
Correctly targeted ES cells were microinjected into blastocysts to produce chimeric mice. Chimerics were bred to C57BL/6J mice. Following germline transmission of the targeted allele, heterozygous mice were interbred to produce wild type (+/+), heterozygous (+/-) and homozygous (-/-) animals. Mice were genotyped by Southern blot analysis. The Exon 6 specific probe hybridized to only a ~16 kb BglI restriction fragment in +/+ mice, a ~11 kb BglI fragment in -/- mice, and to both of these fragments in +/- mice (Fig. 1B).
Mice homozygous for the E2 mutation were born at the expected frequency. Genotype analysis of pups derived from +/- by +/- matings revealed that +/+, +/-, and -/- mice were present at nearly the expected 1:2:1 frequency. Of the initial 60 animals genotyped, 19 (32%) were +/+, 27 (45%) were +/-, and 14 (23%) were -/-. Thus, the E2 gene was dispensable for normal embryonic development. However, as expected, nearly all homozygous mice died in the perinatal period. Immediately following birth, homozygous pups were indistinguishable from their +/+ and +/- littermates; they were vigorous, active and able to suckle. By mid to late day on postnatal day one, most -/- pups became moribund and were readily identifiable as they were lethargic, pale, and exhibited gasping respiratory movements. With few exceptions, -/- pups died within 72 hours of birth. We have observed one rare -/- pup that survived to postnatal day 13. The reason for the prolonged survival of this pup is unknown.
E2 deficient mice accurately model classic MSUD
Immunohistochemistry with an E2 specific antibody was used to examine E2 protein in the mice. As shown in Figure 1C and 1D, immunoreactive E2 protein was abundant in liver and embryonic fibroblasts of+/+ mice. In marked contrast, immunoreactive E2 protein was absent in these same tissues of -/- mice.
Homozygous E2 knockout mice had a nearly 3-fold increase in blood (Figure 2B) and urine (data not shown) levels of BCAA (sum of leucine, isoleucine, and valine) as compared to their +/+ littermates. Because amino acids were analyzed by tandem mass spectrometry, the sum of BCAA shown in Figure 2B also may include alloisoleucine that may have been produced in -/- MSUD mice.
Summary of additional blood amino acid levels in the classic MSUD model and control littermates as determined by tandem mass spectrometry. All samples were collected on the day of birth. All values are mean +/- SEM.
196.1 ± 23.1 (6)
229.0 ± 12.0 (6)
76.8 ± 3.0 (6)
167.6 ± 9.9 (13)
251.0 ± 12.8 (13)
72.3 ± 1.6 (13)
94.6 ± 11.6* (5)
107.8 ± 4.8* (5)
50.4 ± 2.9* (5)
In summary, E2 knockout mice lack BCKDH enzymatic activity, E2 immunoreactivity, and have markedly elevated levels of BCAA in the blood and urine. These metabolic derangements ultimately result in neonatal lethality. These phenotypes are remarkably similar to that observed in humans with the classic form of MSUD. Thus, E2 knockout mice closely model classic MSUD.
Knockout mice expressing a human E2 transgene model a variant form of MSUD that mimics intermediate MSUD
LAP-tTA mice were previously produced and characterized by the Bujard laboratory . Pronuclear microinjection was used to produce transgenic mice that harbored the TRE-E2 transgene. From injections at the University of Pittsburgh, 2 transgene positive founders were identified that led to the generation of 3 different transgenic lines (Lines A, B, & D). From the injections at the University of Cincinnati, we obtained 8 transgene positive founders. Subsequent breeding of these founders revealed that many of the animals had multiple transgene insertion sites that segregated. We established a total of 15 different transgenic lines from these founders. Limited resources allowed us to only focus on a total of 9 of these lines. These lines differed substantially in transgene copy number as compared by Southern blot analysis of tail DNA (data not shown).
Summary of additional blood amino acid levels in the intermediate MSUD model and littermate controls as determined by tandem mass spectrometry. Samples were collected from mice that were 4–6 weeks of age. All values are mean +/- SEM.
225.2 ± 36.6 (11)
106.5 ± 10.5 (11)
67.8 ± 7.6 (11)
175.0 ± 28.7 (10)
68.4 ± 7.7** (10)
58.0 ± 7.0 (10)
144.1 ± 16.9* (10)
65.9 ± 8.9** (10)
37.7 ± 6.5**,*** (10)
Because the LAP-tTA mice that were used to drive expression of transgenic human E2 have been demonstrated to produce liver specific expression , BCKDH enzyme activity and production of human E2 protein in liver of Lines A and 525A was examined. BCKDH enzymatic activity in liver from Lines A and 525A was ~6 and 5%, respectively, of the enzymatic activity present in control liver (Fig. 4C). As shown in Fig. 3B, the amount of human E2 (predicted MW~54 Kd) in these transgenic mice was quite variable between mice. In some of these transgenic mice, the level of human E2 was approximately equal to the amount of mouse E2 (~47 Kd) produced in liver of nontransgenic control mice. The observations that these near normal amounts of E2 protein result in only ~5–6% of normal BCKDH enzyme activity suggest that transgene derived E2 is functioning at a suboptimal level. It is probable that the c-myc tag at the carboxy terminus of the transgene derived human E2 interfered with enzymatic activity. This interpretation is consistent with previous studies which have revealed that the carboxy terminus of an analogous subunit of the pyruvate dehydrogenase complex is essential for subunit interactions  and insertion of a Hisx6 tag on the carboxy terminus of an analogous E. coli subunit interfered with normal subunit assembly . It is also possible that human E2 was not fully functional when complexed with mouse E1 and E3 subunits. Human E2 shares ~88% identity to mouse E2 at the amino acid level. We also used western blot analysis to analyze expression of the E2 transgene in brain, kidney and muscle. As shown in Figure 3C, D and 3E respectively, expression of E2 is negligible in those tissues.
Long-term survival of the mice from these two transgenic lines that survived beyond weaning is plotted in Fig. 4D. Although survival data for control animals was not collected, it is readily apparent that survival of the rescue mice was compromised. By 16 weeks, all mice of Line A were moribund and humanely sacrificed, or were found dead in their cage. Survival of Line 525A appeared to be somewhat better. At 20 weeks of age, ~12% of mice (2 of 16) were still alive. These two rare survivors died at 40 and 60 weeks of age.
In summary, these surviving mice had BCAA/alanine ratios that were intermediate between controls and knockouts and they expressed ~5–6% of normal BCKDH enzyme activity in the liver. These phenotypic observations are remarkably similar to the clinical phenotype observed in MSUD patients with the intermediate form of the disease . Thus, these rescue mice represent a very useful model of the intermediate MSUD phenotype.
A major hurdle in developing new treatments for MSUD has been the lack of a practical, accurate animal model of the disease. This hurdle has now been overcome. In this report, we describe the development and characterization of two genetically engineered mouse models that are phenotypically very similar to MSUD patients with the classic and intermediate forms of the disease.
Our model of classic MSUD was created by gene knockout of the E2 subunit of BCKDH. The phenotype of these knockout animals is strikingly similar to humans with classic MSUD. Knockout mice were born at the expected frequency and appeared normal at birth. Within a day of birth and following suckling, blood levels of BCAA were markedly elevated. Concomitantly, levels of the amino acids alanine, glutamate, and glutamine, whose synthesis is linked with normal metabolism of BCAA, were decreased (Table 1). The activity of BCKDH in livers of homozygous knockout mouse pups was undetectable, accounting for the accumulation of unmetabolized BCAA. Immunoreactive E2 protein was absent in liver and fibroblasts of homozygous pups. The phenotypic behavior of homozygous mouse pups resembles symptoms seen in newborn classic MSUD patients. These include signs of neurologic dysfunction such as seizures, stupor, lethargy, loss of motor activity, and respiratory difficulties . These neurologic symptoms may result from reduced levels of glutamate, glutamine, alanine, and other similar neuroactive amino acids, which are considered the culprit for MSUD encephalopathies in patients [1, 37]. Finally, nearly all of the homozygous pups died within 72 hours of birth. This neonatal lethality is likely due to the accumulation of BCAA to neurotoxic levels, ketoacidosis, brain edema, dehydration, and malnutrition as observed in the MSUD calf  and in classic MSUD patients . Heterozygous knockouts were normal and had normal levels of BCAA despite having approximately half of BCKDH enzymatic activity. From the characterization studies completed thus far, the null mutation mouse accurately represents a model of classic MSUD and appears to be a faithful model of the human disease with respect to several biochemical phenotypes .
To create a model of intermediate MSUD, we used a transgenic strategy to express human E2 in the liver of E2 knockout mice. As mentioned above, E2 knockouts without transgene derived E2 die during the early neonatal period. We show that expression of a human E2 transgene in the liver of these knockout mice is able to rescue the neonatal lethality. Many of the rescue mice survived to adulthood. It is interesting to note that in these mouse lines only ~5–6% of normal BCKDH activity in the liver was sufficient to allow survival. We also demonstrated that BCAA levels in blood of these rescue mice were intermediate between controls and knockouts. The hallmarks of intermediate MSUD human patients are persistently increased levels of BCAA and BCKDH activity in the range of 3–30% of normal . Because of the phenotypic similarities of the rescue mice to the human patients, these genetically engineered mice represent a useful small animal model of the intermediate form of MSUD.
The transgenic approach that was used to create the intermediate MSUD model was based on the tetracycline regulated gene switch system that has been used with great success in other studies, for example . The strategy behind our approach was to create mice with an intermediate MSUD phenotype that could be converted to the classic phenotype at the investigator's discretion by turning off the rescuing transgene. However, we were unable to consistently turn off the human E2 expression cassette by the potent tetracycline analogue, doxycycline in either of the two transgenic lines tested (data not shown). The reason these two lines were unresponsive to doxycycline is unknown. It is conceivable that the integration site of the transgenes was not permissive for regulated expression. Screening of additional lines may be needed to find lines that allow for survival and can also be regulated.
The models described in this communication are important advances over the previously described models. An ENU mouse resembling human MSUD was previously created by disrupting the mitochondrial BCAT gene . The BCKDH activity in the liver and muscle of this mouse was normal even though the blood BCAA levels were markedly elevated. While this is an interesting model, it is not a true model of MSUD because the mutation is not in BCKDH and the levels of BCKDH are normal. Furthermore, no case of MSUD has been described attributing this disease to BCAT deficiency. In addition to this mouse model, a cow model of MSUD has also been previously described [17–19]. However, due to practical constraints imposed by such a large animal model and due to the observation of differences between the MSUD cow model and MSUD humans , this model is also of limited utility. In contrast, the murine models described in this report are more valid and appropriate for modeling human MSUD.
The most significant opportunity presented by the MSUD mouse models is to test novel treatments such as gene [38–41] and cell based therapies (e.g., hepatocytes [42, 43] or embryonic stem cells ). Relevant to gene therapy, recent problems with gene therapy in humans highlight the importance and critical need of animal models of genetic diseases for preclinical studies. The testing of gene and cell based therapies on appropriate animal model systems provides a preliminary method of establishing not only the efficacy, but also short- and long-term safety. Success with animal studies is expected to advance such therapeutic approaches and could pave the way for studies in humans. In addition to testing various treatments, these models should also be very useful for investigating the underlying pathophysiologic consequences of the disease. Such studies are very difficult/impossible to do in human patients for obvious ethical reasons, and often must rely on autopsy tissue. Additionally, the intermediate MSUD model may also be useful for studies to test the effect of thiamin. A thiamin-responsive form of MSUD has been described . The phenotype of these patients is heterogeneous and treatment with a wide range of thiamin doses has produced limited success . Recent cell culture studies with MSUD cells have suggested that the thiamin-responsive phenotype is dependent upon the presence of at least one E2 expressing allele [45, 46]. In light of these newer findings, our intermediate MSUD mouse provides a model to test the effect of thiamin with respect to BCKDH activity and blood amino acid levels at the level of the whole animal. Because of recent interest in structural analysis of multienzyme complexes [35, 36, 45, 46], the intermediate MSUD model may provide a valuable resource for studies of structural biology. Lastly, the information and knowledge gained from studies with the MSUD models described here will also be applicable and transferable to other mitochondrial disorders due to defects in multisubunit enzymes.
In summary, this report describes the development and characterization of genetically engineered mouse models of classic as well as intermediate MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease.
- The abbreviations used are:
MSUD, Maple Syrup Urine Disease
branched chain ketoacid dehydrogenase
branched chain amino acids
branched chain aminotransferase
- ES cell:
embryonic stem cell
tetracycline responsive element
mouse embryonic fibroblast.
The authors would like to thank Frank Kist, Jodi Dagget, Edward Mallick, Brian Sloat, and Judith Rodda for expert technical assistance, and Dr. Susan Hutson for the gift of E2 antiserum. This work was supported by National Institutes of Health Grants DK51960, DK57386, DK57956, AA10422, the Scott C. Foster Metabolic Disease Fund, and the MSUD Family Support Group.
- Chuang D, Shih V: Maple Syrup Urine Disease (Branched-Chain Ketoaciduria). The online metabolic & molecular bases of inherited disease. Edited by: Scriver C, Beaudet A, Sly W, Valle D, Childs B, Kinzler K, Vogelstein B. 2001, New York: McGraw-Hill Medical Publishing, 1971-2005. 8Google Scholar
- Marshall L, DiGeorge A: Maple syrup urine disease in the old order Mennonites. Am J Hum Genet. 1981, 33: 139a-Google Scholar
- Yeaman S: The 2-oxo acid dehydrogenase complexes: Recent advances. Biochem J. 1989, 257: 625-632.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoshino M, Koga Y, Yamashita F: A decrease in glycine cleavage activity in the liver of a patient with dihydrolipoyl dehydrogenase deficiency. J Inherit Metab Dis. 1986, 9 (4): 399-400. 10.1007/BF01800493.View ArticlePubMedGoogle Scholar
- Paul H, Adibi S: Role of ATP in the regulation of branched-chin a-keto acid dehydrogenase activity in liver and muscle of fed, fasted, and diabetic rats. J Biol Chem. 1982, 257: 4875-4881.PubMedGoogle Scholar
- Paxton R, Kuntz MJ, Harris R: Phosphorylation sites and inactivation of branched-chain α-keto acid dehydrogenase from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle. Arch Biochem Biophys. 1986, 244: 187-201. 10.1016/0003-9861(86)90108-6.View ArticlePubMedGoogle Scholar
- Peinemann F, Danner DJ: Maple syrup urine disease 1954 to 1993. J Inherit Metab Dis. 1994, 17 (1): 3-15. 10.1007/BF00735389.View ArticlePubMedGoogle Scholar
- Morton DH, Strauss KA, Robinson DL, Puffenberger EG, Kelley RI: Diagnosis and treatment of maple syrup disease: a study of 36 patients. Pediatrics. 2002, 109 (6): 999-1008. 10.1542/peds.109.6.999.View ArticlePubMedGoogle Scholar
- Cox R, Chuang D: Maple syrup urine disease: clinical and molecular genetic considerations. The molecular and genetic basis of neurological disease. Edited by: Rosenberg R, Prusiner S, DiMauro S, Barchi R, Kunkel L. 1993, Boston: Butterworth-Heinemann, 189-207.Google Scholar
- Danner DJ, Davidson ED, Elsas LJ: Thiamine increases the specific activity of human liver branched chain alpha-ketoacid dehydrogenase. Nature. 1975, 254 (5500): 529-530. 10.1038/254529a0.View ArticlePubMedGoogle Scholar
- Khatra BS, Chawla RK, Sewell CW, Rudman D: Distribution of branched-chain alpha-keto acid dehydrogenases in primate tissues. J Clin Invest. 1977, 59 (3): 558-564.View ArticlePubMedPubMed CentralGoogle Scholar
- Suryawan A, Hawes JW, Harris RA, Shimomura Y, Jenkins AE, Hutson SM: A molecular model of human branched-chain amino acid metabolism. Am J Clin Nutr. 1998, 68 (1): 72-81.PubMedGoogle Scholar
- Bodner-Leidecker A, Wendel U, Saudubray JM, Schadewaldt P: Branched-chain L-amino acid metabolism in classical maple syrup urine disease after orthotopic liver transplantation. J Inherit Metab Dis. 2000, 23 (8): 805-818. 10.1023/A:1026708618507.View ArticlePubMedGoogle Scholar
- Netter JC, Cossarizza G, Narcy C, Hubert P, Ogier H, Revillon Y, Rabier D, Saudubray JM: Mid-term outcome of 2 cases with maple syrup urine disease: role of liver transplantation in the treatment. Arch Pediatr. 1994, 1 (8): 730-734.PubMedGoogle Scholar
- Wendel U, Saudubray JM, Bodner A, Schadewaldt P: Liver transplantation in maple syrup urine disease. Eur J Pediatr. 1999, 158 (Suppl 2): S60-64. 10.1007/PL00014324.View ArticlePubMedGoogle Scholar
- Strauss KA, Mazariegos GV, Sindhi R, Squires R, Finegold DN, Vockley G, Robinson DL, Hendrickson C, Virji M, Cropcho L, Puffenberger EG, McGhee W, Seward LM, Morton DH: Elective liver transplantation for the treatment of classical maple syrup urine disease. Am J Transplant. 2006, 6 (3): 557-564. 10.1111/j.1600-6143.2005.01209.x.View ArticlePubMedGoogle Scholar
- Harper PA, Healy PJ, Dennis JA: Ultrastructural findings in maple syrup urine disease in Poll Hereford calves. Acta Neuropathol (Berl). 1986, 71 (3–4): 316-320. 10.1007/BF00688055.View ArticleGoogle Scholar
- Zhang B, Healy P, Zhao Y, Crabb D, Harris R: Premature translation termination of the pre Ela subunit of the branched-chain a-keoacid dehydrogenase as a cause of maple syrup urine disease in polled herford calves. J Biol Chem. 1990, 265: 2425-2427.PubMedGoogle Scholar
- Baird JD, Wojcinski ZW, Wise AP, Godkin MA: Maple syrup urine disease in five Hereford calves in Ontario. Can Vet J. 1987, 28: 505-PubMedPubMed CentralGoogle Scholar
- Wu J-Y, Kao H-J, Li S-C, Stevens R, Hillman S, Millington D, Chen Y-T: ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease. J Clin Invest. 2004, 113: 434-440. 10.1172/JCI200419574.View ArticlePubMedPubMed CentralGoogle Scholar
- Mansour SL, Thomas KR, Deng C, Capecchi MR: Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988, 336: 348-353. 10.1038/336348a0.View ArticlePubMedGoogle Scholar
- Tybulewicz V, Crawford C, Jackson P, Bronson R, Mulligan R: Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell. 1991, 65: 1153-1163. 10.1016/0092-8674(91)90011-M.View ArticlePubMedGoogle Scholar
- Nagy A, Cocza E, Merenties Diaz E, Prideaux VR, Ivanyi E, Markkula M, Rossant J: Embryonic stem cells alone are able to support fetal development in the mouse. Development. 1990, 110: 815-821.PubMedGoogle Scholar
- Homanics GE, Ferguson C, Quinlan JJ, Daggett J, Snyder K, Lagenaur C, Mi ZP, Wang XH, Grayson DR, Firestone LL: Gene knockout of the α6 subunit of the γ-aminobutyric acid type A receptor: Lack of effect on responses to ethanol, pentobarbital, and general anesthetics. Mol Pharmacol. 1997, 51 (4): 588-596.PubMedGoogle Scholar
- Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 1992, 89: 5547-5551.View ArticlePubMedPubMed CentralGoogle Scholar
- Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD: Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA. 1988, 85 (3): 836-840.View ArticlePubMedPubMed CentralGoogle Scholar
- Palmiter RD, Sandgren EP, Avarbock MR, Allen DD, Brinster RL: Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci USA. 1991, 88 (2): 478-482.View ArticlePubMedPubMed CentralGoogle Scholar
- Kozak M: Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986, 44 (2): 283-292. 10.1016/0092-8674(86)90762-2.View ArticlePubMedGoogle Scholar
- Homanics GE: Knockout and knockin mice. Methods for Alcohol-Related Neuroscience Research. Edited by: Liu Y, Lovinger DM. 2002, Boca Raton: CRC Press LLC, 31-60.Google Scholar
- Hutson SM, Berkich D, Drown P, Xu B, Aschner M, LaNoue KF: Role of branched-chain aminotransferase isoenzymes and gabapentin in neurotransmitter metabolism. J Neurochem . 1998, 71 (2): 863-874.View ArticlePubMedGoogle Scholar
- Paul H, Adibi S: Mechanism of increased conversion of branched-chain keto acid dehydrogenase from inactive to active form by a medium-chain fatty acid (octanoate) in skeletal muscle. J Biol Chem. 1992, 267: 11208-11214.PubMedGoogle Scholar
- Snedecor GW: Statistical Methods. 1955, Ames, IA: Iowa State University Press, 4Google Scholar
- Harper A, Miller R, Block K: Branched-chain amino acid metabolism. Annu Rev Nutr. 1984, 4: 409-454. 10.1146/annurev.nu.04.070184.002205.View ArticlePubMedGoogle Scholar
- Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H, Bujard H: Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci USA. 1996, 93 (20): 10933-10938. 10.1073/pnas.93.20.10933.View ArticlePubMedPubMed CentralGoogle Scholar
- Mattevi A, Obmolova G, Schulze E, Kalk KH, Westphal AH, De Kok A, Hol WGJ: Atomic structure of the cubic core of pyruvate dehydrogenase multienzyme complex. Science. 1992, 255: 1544-1550.View ArticlePubMedGoogle Scholar
- Knapp JE, Carroll D, Lawson JE, Ernst SR, Reed LJ, Hackert ML: Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase. Protein Sci. 2000, 9 (1): 37-48.View ArticlePubMedPubMed CentralGoogle Scholar
- Chuang DT, Chuang JL, Wynn RM: Lessons from genetic disorders of branched-chain amino Acid metabolism. J Nutr. 2006, 136 (1): 243S-249S.PubMedGoogle Scholar
- Wang L, Calcedo R, Nichols TC, Bellinger DA, Dillow A, Verma IM, Wilson JM: Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy. Blood . 2005, 105 (8): 3079-3086. 10.1182/blood-2004-10-3867.View ArticlePubMedGoogle Scholar
- Mochizuki S, Mizukami H, Ogura T, Kure S, Ichinohe A, Kojima K, Matsubara Y, Kobayahi E, Okada T, Hoshika A, Ozawa K, Kume A: Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice. Gene Ther. 2004, 11 (13): 1081-1086. 10.1038/sj.gt.3302262.View ArticlePubMedGoogle Scholar
- Mueller GM, McKenzie LR, Homanics GE, Watkins SC, Robbins PD, Paul HS: Complementation of defective leucine decarboxylation in fibroblasts from a maple syrup urine disease patient by retrovirus-mediated gene transfer. Gene Ther. 1995, 2 (7): 461-468.PubMedGoogle Scholar
- Koyata H, Cox R, Chuang D: Stable correction of maple syrup urine disease in cells from a Mennonite patient by retroviral-mediated gene transfer. Biochem J. 1993, 295: 635-639.View ArticlePubMedPubMed CentralGoogle Scholar
- Ambrosino G, Varotto S, Strom SC, Guariso G, Franchin E, Miotto D, Caenazzo L, Basso S, Carraro P, Valente ML, D'Amico D, Zancan L, D'Antiga L: Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1. Cell Transplant. 2005, 14 (2–3): 151-157.View ArticlePubMedGoogle Scholar
- Muraca M, Gerunda G, Neri D, Vilei MT, Granato A, Feltracco P, Meroni M, Giron G, Burlina AB: Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet. 2002, 359: 317-318. 10.1016/S0140-6736(02)07529-3.View ArticlePubMedGoogle Scholar
- Fair JH, Cairns BA, Lapaglia MA, Caballero M, Pleasant WA, Hatada S, Kim HS, Gui T, Pevny L, Meyer AA, Stafford DW, Smithies O, Frelinger JA: Correction of factor IX deficiency in mice by embryonic stem cells differentiated in vitro. Proc Natl Acad Sci USA. 2005, 102 (8): 2958-2963. 10.1073/pnas.0409840102.View ArticlePubMedPubMed CentralGoogle Scholar
- Chuang J, Wynn R, Moss C, Song J, Li J, Awad N, Mandel H, Chuang D: Structural and biochemical basis for novel mutations in homozygous Israeli maple syrup urine disease patients: a proposed mechanism for the thiamin-responsive phenotype. J Biol Chem. 2004, 279 (17): 17792-17800. 10.1074/jbc.M313879200.View ArticlePubMedGoogle Scholar
- Li J, Wynn RM, Machius M, Chuang JL, Karthikeyan S, Tomchick DR, Chuang DT: Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain alpha-keto acid decarboxylase/dehydrogenase. J Biol Chem. 2004, 279 (31): 32968-32978. 10.1074/jbc.M403611200.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/7/33/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.