Human Molecular Genetics, 2000, Vol. 9, No. 11 1699-1707
© 2000 Oxford University Press
Paradoxical influence of acid ß-galactosidase gene dosage on phenotype of the twitcher mouse (genetic galactosylceramidase deficiency)
1Neuroscience Center, 2Departments of Neurology and Psychiatry and 3Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7250, USA and 4INSERM U189, Lyon-Sud School of Medicine and Fondation Gillet-Mérieux, Lyon-Sud Hospital, 69921 Oullins Cedex, France
Received 30 March 2000; Revised and Accepted 3 May 2000.
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ABSTRACT |
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We have cross-bred twitcher mice (galactosylceramidase deficiency) and acid ß-galactosidase knockout mice (GM1 gangliosidosis) and found that the acid ß-galactosidase gene dosage exerts an unexpected and paradoxical influence on the twitcher phenotype. Twitcher mice with an additional complete deficiency of acid ß-galactosidase have the mildest phenotype with the longest lifespan and nearly rescued CNS pathology. In contrast, twitcher mice with a single functional acid ß-galactosidase gene have the most severe disease with the shortest lifespan, despite the fact that GM1 gangliosidosis carrier mice with an otherwise normal genetic background are phenotypically normal. A significant proportion of these galc–/–, bgal+/– mice clinically develop additional extreme hyper-reactivity and generalized seizures not seen in any other genotypes. Consistent with the clinical seizures, widespread neuronal degeneration is present in the galc–/–, bgal+/– mice, most prominently in the CA3 region of the hippocampus. The double knockout mice show a massive accumulation of lactosylceramide in all tissues. The brain inexplicably contains only a half-normal amount of galactosylceramide, which may account for the mild clinical and pathological phenotype. On the other hand, brain psychosine level is increased in all twitcher mice, but galc–/–, bgal+/– mice show a significantly higher level than other genotypes. The reduced galactosylceramide in the brain of the double knockout mice and the significantly higher psychosine in the brain of the galc–/–, bgal+/– mice cannot readily be explained from the genotypes of these mice. These observations are contrary to the expected outcome of Mendelian autosomal recessive single gene disorders and may also be interpreted as that the acid ß-galactosidase gene functions as a modifier gene for the phenotypic expression of genetic galactosylceramidase deficiency.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Two lysosomal ß-galactosidases, galactosylceramidase and acid ß-galactosidase, are involved in normal turnover of cellular constituents with terminal ß-galactoside residues. Galactosylceramidase (GALC, EC 3.2.1.46) catalyzes hydrolysis of galactosylceramide (1), galactosylsphingosine (2), lactosylceramide (3,4) and monogalactosyl diglyceride (5). Genetic deficiency of this enzyme causes globoid cell leukodystrophy (Krabbe disease) (6). Unlike other sphingolipid storage diseases, abnormal accumulation of the primary natural substrate of the deficient enzyme, galactosylceramide, does not occur in the central nervous system (7). However, an abnormal accumulation of psychosine was demonstrated in the brain of human patients with Krabbe disease (8,9). These findings strongly support the hypothesis that psychosine is responsible for the very rapid loss of oligodendrocytes (2). The extensive globoid cell reaction indicates that the impaired catabolism of galactosylceramide is also an important factor in pathogenesis (10,11). The twitcher mouse is a naturally occurring mouse model of human Krabbe disease caused by a mutation in the galactosylceramidase gene (12,13). Psychosine also accumulates in the brain of twitcher mice (14). The other ß-galactosidase, acid ß-galactosidase (BGAL, EC 3.2.1.23), catalyzes hydrolysis of GM1 ganglioside (15), asialo GM1 ganglioside (GA1) (16), lactosylceramide (4) and keratan sulfate (17). Genetic deficiency of this enzyme causes GM1 gangliosidosis (15). In the brains of patients with GM1 gangliosidosis ganglioside GM1 and its asialo derivative GA1 accumulate (18). The genes coding for the two ß-galactosidases share no base or amino acid sequence similarities (19,20). Recently, we and another laboratory independently generated mutant mouse lines with an inactivated mouse bgal locus that are close models of human GM1 gangliosidosis (21,22).
Because of the overlapping substrate specificities of the two ß-galactosidases, no overt accumulation of lactosylceramide occurs in either Krabbe disease or GM1 gangliosidosis. The cross-breeding experiments between the twitcher mouse and the GM1 gangliosidosis mouse described in this report were initiated with the modest aim of confirming our long-standing prediction that simultaneous deficiency of the two lysosomal ß-galactosidases would result in genuine lactosylceramidosis. While this prediction was readily demonstrated to be correct, the cross-bred mice exhibited additional highly complex and totally unexpected phenotypes that seem to defy plausible explanation on the basis of the current knowledge of these two genes, their products and the metabolism of their substrates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Phenotype
Paradoxical influence of bgal gene dosage (Fig 1).
Twitcher mice with a normal genetic background began to show growth retardation from ~15 days, developed progressive neurological symptoms from ~20 days and almost all of them died before 58 days of age. Their average lifespan was 47.9 ± 4.9 days (n = 41). In contrast, twitcher mice with an additional complete acid ß-galactosidase deficiency (the double knockout mice) exhibited by far the mildest phenotype with the longest lifespans among twitchers of all genotypes. Hepatosplenomegaly was evident in the double knockout mice. They continued to gain weight until ~40 days (data not shown). The neurological manifestations were distinctly milder and they could survive up to 94 days of age. In fact, almost all twitcher mice (galc–/–, bgal+/+) died before any of the double knockout mice started dying. Their average survival was 74.3 ± 9.2 days (n = 25). Even more paradoxically, twitcher mice with a single functional bgal gene (galc–/–, bgal+/–) had the most severe phenotype with the shortest lifespan (Fig. 1). Additionally, these galc–/–, bgal+/– mice developed extreme hyper-reactivity and generalized seizures during a relatively narrow time window of 30–35 days. During the period of hyper-reactivity, these mice ran around aimlessly and jumped out of the cage when the cage was merely touched or the top of the cage was opened. Among 46 mice with this genotype, 11 (24%) showed severe growth failure and died during the period 22–27 days. Beyond this period, seven mice (15%) developed seizures and hyper-reactivity between 30 and 35 days. Almost all of them died before 46 days and their average survival was 37.7 ± 7.8 days (n = 46).
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In contrast, the phenotype of the acid ß-galactosidase-deficient (GM1 gangliosidosis) mice did not appear to be influenced by the status of the galc gene, either a normally active pair or a single active galc gene. Acid ß-galactosidase-deficient mice survived up to ~300 days and developed progressive gait disturbance and feeding problems, as previously described (21,23). Although not documented previously, a substantial proportion of affected mice developed generalized seizures after 250 days. The average lifespan of the bgal-deficient mice was identical between those with a normal pair of the galc gene [306.5 ± 33.1 days (n = 29)] and those with a single functional galc gene [307.2 ± 32.8 days (n = 31)] (mean ± SD).
Pathology
Consistent with the paradoxical clinical phenotypic expression of the twitcher mice of different bgal backgrounds, the pathology was much milder in galc–/–, bgal–/– mice than in twitcher mice with the wild-type bgal background (galc–/–, bgal+/+). The clinically most severe galc–/–, bgal+/– mice exhibited a pathology as severe as twitcher mice with additional neuronal degeneration not seen in other genotypes.
Twitcher pathology is almost rescued in the central nervous system (CNS) of the double knockout mice (Fig. 2A).
The double knockout mice, which in principle should have shown a combined pathology of globoid cell leukodystrophy and GM1 gangliosidosis, actually showed only the pathology of GM1 gangliosidosis until ~40 days, consisting of the typical neuronal storage and scattered vacuolated macrophages in the white matter, leptomeninges and perivascular regions. These macrophages did not show the features of the globoid cells characteristic of twitcher mice. Demyelination was essentially absent in the CNS. However, this lack of a twitcher-like pathology was not complete. A few globoid cell-like macrophages containing needle-shaped inclusions were found in the anterior spinal root and the root exit zone in the spinal cord, where a few demyelinated fibers could also be identified at 40 days. In contrast, twitcher mice with the wild-type bgal background developed the full-blown pathology by 40 days. The twitcher pathology slowly developed in older galc–/–, bgal–/– mice. At 77 days scattered globoid cells with demyelination were present in the brainstem fiber tracts and cerebral and cerebellar white matter. No comparison with twitcher mice can be made since galc–/–, bgal+/+ mice do not survive even to 60 days. Even at 94 days the twitcher-like pathology in the double knockout mice was much milder than that seen in the galc–/–, bgal+/+ mice at 35–40 days. Thus, in the double knockout mice the twitcher pathology was essentially rescued until 35–40 days and then developed thereafter at a very slow rate, while the pathological process of GM1 gangliosidosis appeared to proceed as expected.
Rescue of twitcher pathology is limited to the CNS.
The nearly absent twitcher pathology in the double knockout mouse was limited to the CNS. This could be demonstrated most dramatically by a clear demarcation between the CNS and peripheral nervous system (PNS) portions of the trigeminal nerve (Fig. 2B). Infiltration of globoid cells into the CNS portion was rare at 35–40 days but was progressively more common at 77 and 94 days. The extent of pathology in the sciatic nerve of 94-day-old galc–/–, bgal–/– mice was similar to that in 35- to 40-day-old galc–/–, bgal+/+ mice.
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Liver and other visceral organs are severely affected in the double knockout mice (Fig. 2C).
The liver of 94-day-old galc–/–, bgal–/– mice showed numerous macrophage-filled sinusoids compressing the hepatocytes. Similar macrophages were noted in the lymph follicles in the spleen. The renal tubular epithelium was diffusely vacuolated. Finely vacuolated cells were also present within the Bowman capsule in the glomeruli. These vacuoles were PAS-negative on paraffin sections. This severe visceral pathology is unique to the double knockout genotype and clearly reflects the massive lactosylceramide accumulation in these organs.
The galc–/–, bgal+/– mice have the most severe pathology with additional neuronal degeneration.
The characteristic pathology of globoid cell leukodystrophy in the nervous system of twitcher mice with a single remaining functional acid ß-galactosidase gene (galc–/–, bgal+/–) was as severe and as rapidly progressive as in twitcher mice with the normal bgal background (galc–/–, bgal+/+). These mice, however, exhibited additional neuronal pathology that appeared to provide the morphological counterpart of the clinical seizures observed only among the mice of this genotype. At 27 days many dying neurons with deeply eosinophilic cytoplasm and pyknotic nuclei were present diffusely in the cerebral cortex (Fig. 2D). In the hippocampus neuronal degeneration was most pronounced in the CA3 region and then in the subiculum. The CA1 and CA2 regions contained much fewer degenerating neurons. Scattered necrotic cells were also noted in the hypothalamus, amygdala, caudate/putamen and lateral septal nuclei. In the cerebellum neurons with small hyperchromatic round nuclei were scattered in the molecular and granular cell layers. Purkinje cells and the neurons in the deep cerebellar nuclei were generally well preserved. Activated astrocytes and microglia/macrophages were conspicuous in the cerebral cortex, where many dying cells were present, and to a lesser extent in the white matter. Some anterior horn neurons contained small eosinophilic or amphophilic inclusions, but no neuronal degeneration was noted. At 40 days the neuronal pathology was fundamentally similar but there were fewer dying neurons and more ghosts of dead neurons. On 1 µm sections basophilic neuronal inclusions were seen in the reticular formation and anterior horn neurons and in some neurons in the cerebral cortex. Electron microscopic examinations suggested that they were identical to those seen in long surviving twitcher mice (24). The liver and spleen were free of pathology in the galc–/–, bgal+/– mice.
Biochemistry
Expected tissue enzyme activities.
Activities of tissue galactosylceramidase and acid ß-galactosidase were exactly as expected in the six possible genotypes (Fig. 3). There were no significant differences in the control enzyme, N-acetyl ß-hexosaminidase, among these genotypes (data not shown).
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Massive lactosylceramide accumulation.
As predicted from the overlapping substrate specificities of the two lysosomal ß-galactosidases, there was massive accumulation of lactosylceramide in the double knockout mice (galc–/–, bgal–/–) in all tissues examined: brain, kidney, liver and cultured fibroblasts (Fig. 4A and B). Serine loading experiments with cultured fibroblasts indicated a metabolic block in degradation of lactosylceramide in the double knockout mouse (Fig. 4C). In addition, a moderate accumulation of lactosylceramide was observed in the brain and kidney, but not in other tissues, of twitcher mice with a single active bgal gene (Fig. 4B). In view of the apparently normal turnover of lactosylceramide in this genotype, as indicated by the liver lipid analysis, this accumulation is more likely due to the severe pathology and infiltration of macrophages.
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Reduced brain and kidney galactosylceramide in the double knockout mice.
Brain galactosylceramide was determined quantitatively at 20, 30, 40 and 60 days (Fig. 5). Affected twitcher mice and galc–/–, bgal+/– mice did not survive to 60 days and thus data are not available for these genotypes at this age. Galactosylceramide in the brain was essentially normal in all genotypes during this period, with the conspicuous exception of the double knockout mice (galc–/–, bgal–/–). The relatively normal level of brain galactosylceramide in twitcher mice with a moderate decline only toward the terminal stage of the disease has been well documented previously (25). Throughout the developmental stages examined, brain galactosylceramide was significantly reduced to 50–65% of normal in the double knockout mice despite the improved pathology and better preservation of myelin demonstrated histologically. Twitcher mice are known to accumulate large amounts of galactosylceramide in the kidney over the normally minuscule level (26,27). Therefore, kidney galactosylceramide was evaluated only for mice of galc–/– genotype. Consistent with the finding in the brain, the double knockout mice showed significantly less galactosylceramide in the kidney compared with galc–/–, bgal+/+ and galc–/–, bgal+/– mice (data not shown). No difference was noted between the latter two genotypes.
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Significantly higher brain psychosine in galc–/–, bgal+/– mice.
As expected, brain psychosine, determined at 20, 30, 40 and 60 days, was dramatically increased in all mice with the galc–/– (twitcher) genotype (Fig. 6). However, we observed unexpected and potentially significant variations among the different genotypes. At 30 and 40 days brain psychosine levels in twitcher mice with a single active acid ß-galactosidase (galc–/–, bgal+/–) were significantly higher than in twitcher mice with a normal bgal background and the double knockout mice (P < 0.02). On the other hand, at 40 days brain psychosine level in the double knockout mice was lower than in twitcher mice with a normal bgal background at borderline statistical significance (P = 0.05). At 30 days, however, there was no difference in the brain psychosine levels between twitcher mice and double knockout mice. All of the detected psychosine should be galactosylsphingosine, since, from our previous experience with normal mice and various mutant mice, including mice in which galactosylceramide synthase was inactivated, we know that there is no detectable glucosylsphingosine in the mouse brain (24) with the exception of glucosylceramidase-deficient (Gaucher disease) mice (28).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this series of cross-breeding experiments tissue activities of the two ß-galactosidases were exactly as expected in all six possible genotypes, with deficient activities of both enzymes only in the doubly deficient (galc–/–, bgal–/–) mice. We could readily confirm our prediction that genuine lactosylceramidosis should result only when the activities of both of the two lysosomal ß-galactosidases, galactosylceramidase and acid ß-galactosidase, are deficient because they are both active toward lactosylceramide (4). Accumulation of lactosylceramide in the doubly deficient mice is much more conspicuous in visceral organs than in the brain because in these organs the predominant glycosphingolipids that are degraded via lactosylceramide are the globotriaosylceramide/globoside series of compounds, while in the brain gangliosides are predominant and their degradation is blocked upstream at the GM1/GA1 step.
The results of the experiments turned out to be much more exciting than the confirmation of this straightforward prediction. Many of our observations were unexpected and full of apparent contradictions. Based on the conventional wisdom regarding Mendelian single gene diseases, we had anticipated that: (i) mice that are simultaneously deficient in both ß-galactosidases would show a clinical and pathological phenotype that is a combination of the two diseases plus additional lactosylceramidosis and consequently more severe than either of the diseases alone; (ii) heterozygosity of either gene would not affect the phenotypic expression of the other disease. In fact, the galc gene dosage, either null, single active or normally active pair, did not appear to modify the phenotype of GM1 gangliosidosis. It was the effects of bgal gene dosage on phenotypic expression of twitcher mice that appeared to defy these conventional predictions. The essence of the puzzle is that twitcher mice exhibit the most severe clinical and pathological phenotype when they retain a single normally active bgal gene, while twitcher mice with a pair of normally active bgal genes have an intermediate phenotype and those that are also completely deficient in acid ß-galactosidase have by far the mildest disease. It is important to note that none of the substrates of either enzyme are serially located on the same degradative pathway, with the sole exception of lactosylceramide, which is degraded by both enzymes, and thus genetic deficiency of the other enzyme should not affect their metabolism.
Despite the additional massive accumulation of lactosylceramide in many tissues, the mice doubly deficient in both GALC and BGAL (galc–/–, bgal–/–) had a distinctly milder disease with milder neurological signs, slower progression and a much longer lifespan than twitcher mice with a normal bgal background. The pathology of the visceral organs, which is non-existent in twitcher mice and only mild in GM1 gangliosidosis, was extensive in the doubly deficient mice, reflecting lactosylceramide accumulation. Consistent with the milder neurological phenotype, the characteristic twitcher pathology was nearly rescued in the CNS in the early stages, although it did gradually develop in later stages. The additional complication is that the much milder pathology was confined to the CNS and that the peripheral nerve pathology appeared as severe as in twitcher mice with normal BGAL. We are unable to offer explanations for this surprising outcome beyond a few speculations. We searched for clues in biochemistry. Since galactosylsphingosine (psychosine), rather than galactosylceramide, is considered primarily responsible for the devastating neuropathology in twitcher mice and other mammalian globoid cell leukodystrophies, including human Krabbe disease, we thought that psychosine accumulation might be even greater when both lysosomal ß-galactosidases were absent. The analytical results clearly excluded this possibility. The brain psychosine levels were similar in twitcher mice with a normal bgal background and in those with totally deficient BGAL activity. Biochemically, the brain of doubly deficient mice showed three abnormalities; accumulation of lactosylceramide as the consequence of double deficiency, accumulation of GM1 ganglioside as the consequence of BGAL deficiency and a reduction in galactosylceramide to half the normal value. We are not aware of any experimental data, or in fact any suggestion, that increased lactosylceramide might have beneficial effects on any type of pathology of the brain. On the other hand, there is voluminous literature suggesting beneficial effects of GM1 ganglioside on protection from damage and/or promotion of regeneration of once damaged brain tissues, although its underlying mechanism has never been clarified. While we cannot rigorously exclude this possibility as the underlying mechanism of the mild pathology, two factors seem somewhat against it. The primary accumulation of GM1 ganglioside occurs in neuronal perikarya and not in the oligodendrocytes, which is the primary site of globoid cell leukodystrophy pathology. Secondly, GM1 ganglioside exogenously added to cultured skin fibroblasts failed to protect them from apoptosis induced by either C-6 ceramide or psychosine (unpublished results), although such an effect has been described in the literature (29,30). From the genomic manipulation of the doubly deficient mice it is difficult to understand why galactosylceramide levels are reduced to half the normal levels. The doubly deficient mice have more myelin than twitcher mice with a normal bgal background and thus, if anything, one would expect more galactosylceramide than in twitcher mice. As an incidental conclusion, the double knockout mice definitively excluded the hypothesis proposed on the basis of in vitro experiments that galactosylceramide accumulation does not occur in globoid cell leukodystrophy because it can also be hydrolyzed to some extent by BGAL (31). However, whatever the underlying metabolic mechanism of the reduced galactosylceramide may be, the reduced amount of galactosylceramide could be a significant factor in the milder phenotype in the doubly deficient mice. Radin first proposed alleviating the burden of degradation by restricting synthesis of the substrate (32,33). This approach has attracted increasing interest in recent years because of the encouraging positive outcome observed using experimentally generated mouse models of Tay–Sachs disease and Sandhoff disease (34–36). We also observed an improvement in the clinical and pathological phenotype of twitcher mice which had only a single active galactosylceramide synthase gene (37). In the latter case the reduction in galactosylceramide synthesis was to approximately two-thirds of normal and yet the mice showed detectable phenotypic improvements. In the galc–/–, bgal–/– mice the reduction in galactosylceramide was greater and the phenotypic improvement was even more impressive. There are still other possibilities to explain the milder phenotype. For example, if acid ß-galactosidase is required to develop the characteristic twitcher pathology, particularly globoid cell infiltration, the doubly deficient mice will not develop the pathology in the absence of GALC activity. Matsushima et al. (38) described a milder twitcher phenotype when the mice were also deficient in a MHC complex. Recently, Schmid et al. (39) also reported that mice deficient in myelin protein P0 show better preservation of myelin when they are also immune deficient. The precise mechanisms of interaction between the pathology of the nervous system and the immune system in these double knockout mice are not clear.
Twitcher mice (galc–/–) that are simultaneously heterozygous carriers for GM1 gangliosidosis (bgal+/–) unexpectedly had the most severe disease with the shortest lifespan. They exhibited generalized seizures at ~30–35 days which are not seen in other genotypes. Perhaps reflecting these clinical seizures, neuronal degeneration was observed most prominently in the cerebral cortex and the CA3 region of the hippocampus in these mice. This was unexpected because heterozygous carrier mice for GM1 gangliosidosis are phenotypically completely normal. The only biochemical finding that appears to be consistent with this severe phenotype is the clearly increased amount of brain psychosine in this genotype compared with twitcher mice of other bgal genotypes. However, this is unlikely to be the cause of the neuronal degeneration, because psychosine is synthesized by UDP-galactose:ceramide galactosyltransferase, expression of which is limited to myelinating cells, oligodendrocytes and Schwann cells in the nervous system and thus its accumulation is not expected in neurons. The only substrate known to be shared between the two ß-galactosidases is lactosylceramide. In twitcher mice with a single dose of active bgal the normal four gene dosage is reduced to one for its degradation and in fact some accumulation of lactosylceramide was observed in the brain. However, this is also unlikely to account for the neuronal degeneration and the severe phenotype because the doubly deficient mice have much more conspicuous lactosylceramide accumulation in the total absence of the degradation mechanism and yet they have the mildest disease, without neuronal involvement.
Our observations on these hybrid mice not only present an intriguing puzzle as to the metabolic inter-relationship between these two specific gene products and the respective diseases caused by their genetic defects but also have more general, wider implications. The only clearly known difference in the metabolism of the substrates of these two ß-galactosidases between the mouse and humans is that in the mouse GM1 ganglioside is readily desialylated to its asialo derivative, GA1, while this pathway is essentially inactive in humans (21). This difference should not pose an obstacle to extrapolating our results on the mouse to humans. Generally stated, our findings indicate that in an autosomal recessive Mendelian single gene disorder the status of another gene not directly related to the disease can dramatically and in unexpected ways modify the phenotype of the disease. This possibility must always be kept in mind in our consideration of genotype/phenotype correlations. The highly complex genetic background of human patients further compounds the uncertainty. There are well-documented cases of human Krabbe disease, the genetic equivalent of the twitcher mouse, where sibs with identical galc mutations exhibit dramatically distinct clinical phenotypes. It is also well known that identical mutations in the same family can result in an affected sib with cerebral adrenoleukodystrophy and another with adrenomyeloneuropathy without cerebral manifestations. Similar phenomena are known in many other genetic disorders. These considerations lead to the notion that in the cross-breeding experiments between the two mouse mutants we may be observing a specific example of a ‘modifier gene’ at work.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Nomenclature
In this article the mouse genes encoding galactosylceramidase and acid ß-galactosidase are written in lower case italic (galc, bgal) and their products (enzyme proteins) in upper case (GALC, BGAL). Although the twitcher mutation should strictly be expressed as galctwi/twi, the designation galc–/– is used to denote the twitcher mutant, since there is no ambiguity.
Generation of the double mutant of both ß-galactosidases
The twitcher mice are on the inbred C57BL/6J background. The acid ß-galactosidase-deficient mouse line was generated through the gene targeting technique by disrupting exon 6 of the acid ß-galactosidase gene (bgal) with the neomycin resistance (neo) gene (21). While the initial mutants were necessarily on a mixed genetic background, they were back-crossed several generations to the C57BL/6J background. Mice heterozygous for the twitcher mutation (galc+/–) and for disrupted acid ß-galactosidase (bgal+/–) were mated to generate double heterozygous mice (galc+/–, bgal+/–). Finally, the double heterozygous mice were mated with each other to generate doubly deficient mice. In order to more efficiently generate twitcher mice with either two normal bgal genes or a single active bgal gene, we also bred the double heterozygous mice with galc+/–, bgal+/+ mice. Comparisons were made only among the offspring of the double heterozygote mating and the double heterozygote and galc+/– mating to minimize possible influence of the genetic background.
The genotypes of mice were determined on genomic DNA extracted from the clipped tail tip at post-natal day 12 or earlier. The genotype of twitcher mice was diagnosed by a PCR procedure (13). Sakai et al. (13) did not specify the reaction mixture; however, the following conditions gave consistent and reliable results in our hands. The PCR was performed in a total volume of 50 µl containing 0.4 µM each primer, 0.2 mM dNTPs, 1.5 U Taq DNA polymerase and 100–200 ng genomic DNA in 60 mM Tris–HCl, pH 9.5, 15 mM (NH4)2SO4, 3.5 mM MgCl2 and 4% DMSO. Samples were denatured for the first cycle at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, 57°C for 1 min and 72°C for 1 min, with a final extension reaction at 72°C for 7 min. The genotype of acid ß-galactosidase-deficient mice was determined by PCR using two primers both located within exon 6 of the mouse acid ß-galactosidase gene (sense, 5'-GTTGAGATTGAGTACGGGTCCT-3'; antisense, 5'-CTGTTCCAAAATCCACTGTGGC-3') and another pair of primers within the neo gene (sense, 5'-GGAGAGGCTATTCGGCTATGAC-3'; antisense, 5'-CGCATTGCATCAGCCATGATGG-3') (40), because the neo gene was inserted at an AatII site within exon 6 of the acid ß-galactosidase gene. Reaction products were analyzed by electrophoresis on 1.2% agarose gels. The neo primers, if positive, gave a 315 bp fragment and the bgal primers for the wild-type gene gave a 181 bp fragment. Thus, wild-type mice gave only the 181 bp fragment, whereas heterozygous mice gave both the 181 and 315 bp fragments and acid ß-galactosidase-deficient mice had only the 315 bp fragment. Combination of these PCR-based procedures allowed unequivocal diagnosis of all of the nine possible genotypes with respect to the galc and bgal genes.
Clinical studies
All mice were closely observed throughout their lives. Body weight was recorded daily as an objective parameter for development and progression of the disease. In order to determine the natural course of the disease, some mice were allowed to live as long as they could be maintained humanely according to the acceptable practice of laboratory animal care but without forced feeding, intubation or other extraneous interventions. For biochemical and pathological evaluation some mice were killed at 20, 30, 40, 60 and 300 days. A few long-surviving double knockout mice were also included at ages when they had to be killed.
Enzymic assay
Assays for galactosylceramidase and acid ß-galactosidase were done on the brain of 40-day-old mice of each genotype. Freshly obtained brains were homogenized with double distilled water (20% w/v). The protein content was determined by a modified Lowry method using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Activity of GALC was determined with tritium-labeled galactosylceremide as substrate (41). Activity of acid ß-galactosidase was determined with 4-methylumbelliferyl-ß-galactoside (Sigma, St Louis, MO). As a control, activities of total ß-hexosaminidase were measured with 4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide.
Histopathology
The mice were anesthetized with ether, perfused with 4% paraformaldehyde in 0.1 M sodium phosphate, pH 7.4, and immersed in the same fixative at 4°C overnight. Then the brain, optic nerve, spinal cord, trigeminal nerves and ganglia, sciatic nerves, liver, spleen and kidney were removed. Tissues were processed for paraffin and plastic embedding, sectioned and analyzed by light and electron microscopy as previously described (42). The paraffin sections of the CNS and PNS tissues were stained with solochrome and eosin and with luxol fast blue/periodic acid Schiff (PAS). The systemic organs were stained with hematoxylin and eosin and with PAS. Portions of brains were placed in 30% sucrose in 0.1 M sodium phosphate buffer, pH 7.4, for 2 days and then embedded in OCT tissue freezing medium (Sakura Fintek, Forrance, CA) by rapid freezing in Histo-Bath (Shandon Lipshaw, Pittsburgh, PA). Frozen sections of the CNS tissues were cut and stained with PAS.
Lipid extraction from tissues
The tissue was homogenized with water at a 20% concentration by weight in an all-glass Potter–Elvehjem homogenizer. Initial extraction with chloroform/methanol was done as described earlier (43). Brain and kidney lipids were fractionated to neutral and acidic fractions using a reverse phase column essentially according to Kyrklund et al. (44) (Varian Bond Elute, C-18, 3 ml/500 mg). This procedure has the advantages of clean separation of all gangliosides and sulfatides into the acidic fraction, simple and rapid desalting and no need for saponification of the acidic fraction. For analysis of liver lipids, 500 µl of 20% liver homogenates were extracted by addition of 3 ml of chloroform/methanol (1:1 v/v). After at least 15 min the extract was centrifuged at 1000 g for 10 min and the supernatant transferred to a small round-bottom flask. The pellet was resuspended in 3 ml of chloroform/methanol (1:1 v/v). After centrifugation the combined supernatants were dried under nitrogen gas and the lipids were dissolved in 3 ml of chloroform/methanol/water (60:30:4.5 by vol). The extract was desalted through a 1 g Sephadex G-25 Fine column (Pharmacia). For liver samples ganglioside and non-ganglioside fractions were separated by the conventional solvent partitioning procedure.
Removal of glycerophospholipids from the non-ganglioside lipid fraction
Aliquots of the brain neutral lipid fraction and the liver non-ganglioside fraction were subjected to the mercuric chloride saponification procedure in order to remove essentially all glycerophospholipids (45).
Thin layer chromatography and quantitation of lipids
Thin layer chromatography and quantitation of tissue lipids, except for psychosine, were done with Merck high performance TLC plates, appropriate solvents and sprays for separation and visualization and a CAMAG scanning densitometer as described previously (43).
Quantitation of psychosine
Brain psychosine (galactosylsphingosine) was determined by a HPLC procedure (46,47) as modified by us more recently (48), except that the mobile phase was methanol/5 mM sodium phosphate buffer, pH 7.0 (89:11) with 50 mg/l sodium octylsulfate as ion pairing agent (24,37). The peaks of phytosphingosine, psychosine (galactosylsphingosine), sphingosine, sphinganine and eicosasphinganine (internal standard) were eluted in that order and were cleanly separated from each other and from other interfering fluorescent materials. Glucosylsphingosine and galactosylsphingosine were eluted with the same retention time in this HPLC system. The tissue levels of psychosine corrected for the internal standard and the relative detector response were expressed in pmol/mg tissue protein. The data were evaluated by Student’s t-test.
Serine loading experiment with cultured fibroblasts
The serine loading test to evaluate overall sphingolipid metabolism was done with cultured embryonic fibroblasts essentially according to previously published procedures (49).
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ACKNOWLEDGEMENTS |
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This work was presented in part at the 49th annual meeting of the American Society of Human Genetics and published in abstract form [J. Tohyama, M.T. Vanier, K. Suzuki, T. Ezoe, J. Matsuda, and K. Suzuki (1999) Paradoxical effect of acid ß-galactosidase gene dosage on the phenotype of twitcher mouse (genetic galactosylceramidase deficiency). Am. J. Hum. Genet., 65 (suppl.), A96]. The authors thank Ms Elise Cash, Ms Tami Lee, Ms Clarita Langaman and Ms Shin-ja Kim for the tissue preparation and Mr Joe Langaman for cell culture and other technical assistance. This work was supported in part by research grant RO1-NS24289, a Mental Retardation Research Center Core Grant P30-HD03110 from the USPHS and a research grant 83A from the Mizutani Foundation to Kunihiko Suzuki, grant RO1-NS24453 to Kinuko Suzuki and a research grant from the European Leukodystrophy Association (ELA) to M.T.V.
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FOOTNOTES |
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+ Present address: National Nishi-Niigata Hospital, Masago, Niigata 950-2085, Japan
§ Present address: Tokyo Metropolitan Higashi-yamato Medical Center for the Handicapped, Higashi-yamato, Tokyo 207-0022, Japan
¶ To whom correspondence should be addressed at: Neuroscience Center, CB 7250, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7250, USA. Tel: +1 919 966 2405; Fax: +1 919 966 1322; Email: kuni.suzuki@attglobal.net
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Bowen, D. and Radin, N.S. (1969) Cerebroside galactosidase: a method for determination and a comparison with other lysosomal enzymes in developing rat brain. J. Neurochem., 16, 501–511.[Web of Science][Medline]
2 Miyatake, T. and Suzuki, K. (1972) Globoid cell leukodystrophy: additional deficiency of psychosine galactosidase. Biochem. Biophys. Res. Commun., 48, 538–543.[Web of Science]
3 Wenger, D.A., Sattler, M. and Hiatt, W. (1974) Globoid cell leukodystrophy: deficiency of lactosyl ceramide beta-galactosidase. Proc. Natl Acad. Sci. USA, 71, 854–857.
4 Tanaka, H. and Suzuki, K. (1975) Lactosylceramide ß-galactosidase in human sphingolipidoses: evidence for two genetically distinct enzymes. J. Biol. Chem., 250, 2324–2332.
5 Wenger, D.A., Sattler, M. and Markey, J.P. (1973) Deficiency of monogalactosyl diglyceride ß-galactosidase activity in Krabbe’s disease. Biochem. Biophys. Res. Commun., 53, 680–685.[Web of Science][Medline]
6 Suzuki, K. and Suzuki, Y. (1970) Globoid cell leucodystrophy (Krabbe’s disease): deficiency of galactocerebroside ß-galactosidase. Proc. Natl Acad. Sci. USA, 66, 302–309.
7 Suzuki, K., Suzuki, Y. and Suzuki, K. (1995) In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York, NY, pp. 2671–2692.
8 Vanier, M.-T. and Svennerholm, L. (1976) In Volk, B.W. and Schneck, L. (eds), Current Trends in Sphingolipidoses and Allied Disorders. Plenum Press, New York, NY, Vol. 68, pp. 115–126.
9 Svennerholm, L., Vanier, M.-T. and Månsson, J.-E. (1980) Krabbe disease: a galactosylsphingosine (psychosine) lipidosis. J. Lipid Res., 21, 53–64.[Abstract]
10 Austin, J.H. and Lehfeldt, D. (1965) Studies in globoid (Krabbe) leukodystrophy. III. Significance of experimentally-produced globoid-like elements in rat white matter and spleen. J. Neuropathol. Exp. Neurol., 24, 265–289.[Web of Science][Medline]
11 Suzuki, K., Tanaka, H. and Suzuki, K. (1976) In Volk, B.W. and Schneck, L. (eds), Current Trends in Sphingolipidoses and Allied Disorders. Plenum Press, New York, NY, pp. 99–113.
12 Kobayashi, T., Yamanaka, T., Jacobs, J.M., Teixeira, F. and Suzuki, K. (1980) The twitcher mouse: an enzymatically authentic model of human globoid cell leukodystrophy (Krabbe disease). Brain Res., 202, 479–483.[Web of Science][Medline]
13 Sakai, N., Inui, K., Tatsumi, N., Fukushima, H., Nishigaki, T., Taniike, M., Nishimoto, J., Tsukamoto, H., Yanagihara, I., Ozone, K. and Okada, S. (1996) Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe’s disease. J. Neurochem., 66, 1118–1124.[Web of Science][Medline]
14 Igisu, H. and Suzuki, K. (1984) Progressive accumulation of toxic metabolite in a genetic leukodystrophy. Science, 224, 753–755.
15 Okada, S. and O’Brien, J.S. (1968) Generalized gangliosidosis: beta-galactosidase deficiency. Science, 160, 1002–1004.
16 Tanaka, H. and Suzuki, K. (1977) Sustrate specificities of the two genetically distinct human brain ß-galactosidases. Brain Res., 122, 325–335.[Web of Science][Medline]
17 Suzuki, K. (1968) Cerebral GM1-gangliosidosis: chemical pathology of visceral organs. Science, 159, 1471–1472.
18 Suzuki, Y., Sakuraba, H. and Oshima, A. (1995) In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York, NY, pp. 2785–2823.
19 Chen, Y.Q., Rafi, M.A., De Gala, G. and Wenger, D.A. (1993) Cloning and expression of cDNA encoding human galactocerebrosidase, the enzyme deficient in globoid cell leukodystrophy. Hum. Mol. Genet., 2, 1841–1845.
20 Oshima, A., Tsuji, A., Nagano, Y., Sakuraba, H. and Suzuki, Y. (1988) Cloning, sequencing and expression of cDNA for human beta-galactosidase. Biochem. Biophys. Res. Commun., 157, 238–244.[Web of Science][Medline]
21 Hahn, C.N., Martin, M.D., Schröder, M., Vanier, M.T., Hara, Y., Suzuki, K. and d’Azzo, A. (1997) Generalized CNS disease and massive GM1-ganglioside accumulation in mice defective in lysosomal acid ß-galactosidase. Hum. Mol. Genet., 6, 205–211.
22 Matsuda, J., Suzuki, O., Oshima, A., Ogura, A., Noguchi, Y., Yamamoto, Y., Asano, T., Takimoto, K., Sukegawa, K., Suzuki, Y. and Naiki, M. (1997) ß-Galactosidase-deficient mouse as an animal model for GM1-gangliosidosis. Glycoconjugate J., 14, 729–736.[Web of Science][Medline]
23 Matsuda, J., Suzuki, O., Oshima, A., Ogura, A., Naiki, M. and Suzuki, Y. (1997) Neurological manifestations of knockout mice with ß-galactosidase deficiency. Brain Dev., 19, 19–20.[Web of Science][Medline]
24 Ezoe, T., Vanier, M.T., Oya, Y., Popko, B., Tohyama, J., Matsuda, J., Suzuki, K. and Suzuki, K. (2000) Biochemistry and neuropathology of mice doubly deficient in synthesis and degradation of galactosylceramide. J. Neurosci. Res., 59, 170–178.[Web of Science][Medline]
25 Igisu, H., Shimomura, K., Kishimoto, Y. and Suzuki, K. (1983) Lipids of developing brain of twitcher mouse—an authentic murine model of human Krabbe disease. Brain, 106, 405–417.
26 Ida, H., Umezawa, F., Kasai, E., Eto, Y. and Maekawa, K. (1982) An accumulation of galactocerebroside in kidney from mouse globoid cell leukodystrophy (twitcher). Biochem. Biophys. Res. Commun., 109, 634–638.[Web of Science][Medline]
27 Igisu, H., Takahashi, H., Suzuki, K. and Suzuki, K. (1983) Abnormal accumulation of galactosylceramide in the kidney of twitcher mouse. Biochem. Biophys. Res. Commun., 110, 940–944.[Web of Science][Medline]
28 Orvisky, E., McKinney, C.E., Krasnewich, D., Eliason, W.K., Tayebi, N., Martin, B.M., Ginns, E.I. and Sidransky, E. (1997) Glucosylsphingosine accumulation in the brain, liver and spleen of type 2 Gaucher mice. Am. J. Hum. Genet., 61, A258.
29 Ferrari, G., Anderson, B.L., Stephens, R.M., Kaplan, D.R. and Greene, L.A. (1995) Prevention of apoptotic neuronal death by GM1 ganglioside. Involvement of Trk neurotrophin receptors. J. Biol. Chem., 270, 3074–3080.
30 Cavallini, L., Venerando, R., Miotto, G. and Alexandre, A. (1999) Ganglioside GM1 protection from apoptosis of rat heart fibroblasts. Arch. Biochem. Biophys., 370, 156–162.[Web of Science][Medline]
31 Kobayashi, T., Shinnoh, N., Goto, I. and Kuroiwa, Y. (1985) Hydrolysis of galactosylceramide is catalyzed by two genetically distinct acid beta-galactosidases. J. Biol. Chem., 260, 14982–14987.
32 Warren, K.R., Misra, R.S., Arora, R.C. and Radin, N.S. (1976) Glycosyltransferases of rat brain that make cerebrosides: substrate specificity, inhibitors and abnormal products. J. Neurochem., 26, 1063–1072.[Web of Science][Medline]
33 Radin, N.S. (1999) Chemotherapy by slowing glucosphingolipid synthesis. Biochem. Pharmacol., 57, 589–595.[Web of Science][Medline]
34 Platt, F.M., Neises, G.R., Reinkensmeier, G., Townsend, M.J., Perry, V.H., Proia, R.L., Winchester, B., Dwek, R.A. and Butters, T.D. (1997) Prevention of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin. Science, 276, 428–431.
35 Platt, F.M. and Butters, T.D. (1998) New therapeutic prospects for the glycosphingolipid lysosomal storage diseases. Biochem. Pharmacol., 56, 421–430.[Web of Science][Medline]
36 Jeyakumar, M., Butters, T.D., Cortina-Borja, M., Hunnam, V., Proia, R.L., Perry, V.H., Dwek, R.A. and Platt, F.M. (1999) Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin. Proc. Natl Acad. Sci. USA, 96, 6388–6393.
37 Ezoe, T., Vanier, M.T., Oya, Y., Popko, B., Tohyama, J., Matsuda, J., Suzuki, K. and Suzuki, K. (2000) Twitcher mice with only a single active galactosylceramide synthase gene exhibit clearly detectable but therapeutically minor phenotypic improvements. J. Neurosci. Res., 59, 179–187.[Web of Science][Medline]
38 Matsushima, G.K., Taniike, M., Glimcher, L.H., Grusby, M.J., Frelinger, J.A., Suzuki, K. and Ting, J.P.Y. (1994) Absence of MHC class II molecules reduces CNS demyelination, microglial macrophage infiltration and twitching in murine globoid cell leukodystrophy. Cell, 78, 645–656.[Web of Science][Medline]
39 Schmid, C.D., Stienekemeier, M., Oehen, S., Bootz, F., Zielasek, J., Gold, R., Toyka, K.V., Schachner, M. and Martini, R. (2000) Immune deficiency in mouse models for inherited peripheral neuropathies leads to improved myelin maintenance. J. Neurosci., 20, 729–735.
40 Tybulewicz, V.L., Crawford, C.E., Jackson, P.K., Bronson, R.T. and Mulligan, R.C. (1991) Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell, 65, 1153–1163.[Web of Science][Medline]
41 Suzuki, K. (1987) Enzymatic diagnosis of sphingolipidoses. Methods Enzymol., 138, 727–762.[Web of Science][Medline]
42 Oya, Y., Nakayasu, H., Fujita, N., Suzuki, K. and Suzuki, K. (1998) Pathological study of mice with total deficiency of sphingolipid activator proteins (SAP knockout mice). Acta Neuropathol., 96, 29–40.[Medline]
43 Fujita, N., Suzuki, K., Vanier, M.T., Popko, B., Maeda, N., Klein, A., Henseler, M., Sandhoff, K., Nakayasu, H. and Suzuki, K. (1996) Targeted disruption of the mouse sphingolipid activator protein gene: a complex phenotype, including severe leukodystrophy and wide-spread storage of multiple sphingolipids. Hum. Mol. Genet., 5, 711–725.
44 Kyrklund, T. (1987) Two procedures to remove polar contaminants from a crude brain lipid extract by using prepacked reverse-phase columns. Lipids, 22, 274–277.[Web of Science][Medline]
45 Abramson, M.B., Norton, W.T. and Katzman, R. (1965) Study of ionic structure in phospholipids by infrared spectra. J. Biol. Chem., 240, 2389–2395.
46 Merrill Jr, A.H., Wang, E., Mullins, R.E., Jamison, W.C., Nimkar, S. and Liotta, D.C. (1988) Quantitation of free sphingosine in liver by high-performance liquid chromatography. Anal. Biochem., 171, 373–381.[Web of Science][Medline]
47 Rodriguez-Lafrasse, C., Rousson, R., Pentchev, P.G., Louisot, P. and Vanier, M.T. (1994) Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders. Biochim. Biophys. Acta, 1226, 138–144.[Medline]
48 Matsumoto, A., Vanier, M.T., Oya, Y., Kelly, D., Popko, B., Wenger, D.A., Suzuki, K. and Suzuki, K. (1997) Minimal increment in galactosylceramidase expression is sufficient for significant phenotypic improvement in twitcher mouse. Dev. Brain Dysfunct., 10, 142–154.
49 Klein, A., Henseler, M., Klein, C., Suzuki, K., Harzer, K. and Sandhoff, K. (1994) Sphingolipid activator protein D (sap-D) stimulates the lysosomal degradation of ceramide in vivo. Biochem. Biophys. Res. Commun., 200, 1440–1448.[Web of Science][Medline]
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