Human Molecular Genetics, 2001, Vol. 10, No. 11 1191-1199
© 2001 Oxford University Press
A mutation in the saposin A domain of the sphingolipid activator protein (prosaposin) gene results in a late-onset, chronic form of globoid cell leukodystrophy in the mouse
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 U 189, Lyon-Sud School of Medicine and Fondation Gillet-Mérieux, Lyon-Sud Hospital, 69921 Oullins Cedex, France
Received 14 February 2001; Revised and Accepted 30 March 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins derived from a common precursor protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A–/– mice developed slowly progressive hind leg paralysis with clinical onset at
2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Sphingolipid activator proteins (saposins A, B, C and D) are small heat-stable glycoproteins required for in vivo degradation of some sphingolipids with short carbohydrate chains (1). They are derived from a common precursor protein (prosaposin), which is encoded by a single gene, and proteolytically processed to saposins A, B, C and D. These four saposins are all homologous to each other, having six conserved cysteines and one common glycosylation site. Inspite of these structural similarities, their activator functions are specific, with some overlaps, for individual sphingolipid hydrolases. Human patients with mutations in the saposin B and C domains are known and they show phenotypes of metachromatic leukodystrophy and Gaucher disease, indicating that their primary in vivo substrates are sulfatide and glucosylceramide, respectively (2,3). Two mutations are known in humans which result in complete inactivation of all four saposins and prosaposin (4,5). In addition, we previously generated a mouse model of total saposin deficiency with the gene-targeting technology that closely mimics the human disease (6). Total saposin deficiency, both in humans and mice, is a devastating disease with a complex phenotype involving multiple organs and multiple sphingolipids, indicating the essential roles of these saposins in vivo. There are reports suggestive of saposin A being a galactosylceramidase (GALC) activator (7,8), and saposin D being a ceramidase activator (9,10). However, absence of specific genetic deficiency of either saposin A or D leaves unanswered the question of whether either saposin A or D is indispensable for normal cellular function. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system (11) which eliminated one of the three disulfide bonds. The maintenance of the three strictly conserved disulfide bridges is considered essential for the functional properties of saposins (12). In humans, an equivalent mutation in the 4th cysteine to phenylalanine in saposin C causes specific saposin C deficiency, and a mutation of the 5th cysteine to serine in saposin B causes specific saposin B deficiency (1). In these human disorders of saposin B and C deficiency, the other unaffected saposins are properly processed and fully functional. The saposin A–/– mice developed a late-onset, chronic form of globoid cell leukodystrophy (GLD), clearly indicating that saposin A is indispensable for GALC to degrade galactosylceramide (GalCer) in vivo and that genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy, without GALC deficiency.
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RESULTS |
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Gene targeting and clinical phenotype
The targeting vector was designed to introduce a mutation in exon 4 that changed the 4th cysteine in saposin A to phenylalanine, resulting in destruction of one of the disulfide bridges in saposin A (Fig. 1). Correctly targeted embryonic stem (ES) cells, before and after removal of the neomycin resistance gene (Neo) by Cre recombinase, and genotypes of resultant mice were confirmed by appropriate DNA analyses (Fig. 2A and B). The allele with the C106F mutation in the saposin A domain generated stable prosaposin mRNA of normal length, as expected, in contrast to its absence in the total saposin deficiency (Fig. 2C). Viable saposin A–/– mice were obtained with the frequency expected from the Mendelian principle. Saposin A–/– mice initially grew normally and were indistinguishable from their littermates. However, at
2.5 months, careful observation could distinguish affected mice from littermates by their subtle hind leg weakness and sluggish activities. Paralysis and atrophy of hind legs progressed slowly and the mice stopped gaining weight at 3 months (Fig. 3A). Some of the saposin A–/– mice showed generalized seizures and hyperactivity at 3 months. Twitching, prominently seen in twitcher mice and other myelin mutants, did not become obvious until the end stage. Both males and females were fertile, and females were able to raise their offspring normally at least twice. Ileus and neurogenic bladder, reflecting autonomic nervous system dysfunction, were commonly seen, as is also the case in human adult-onset leukodystrophy. Gait disturbance, feeding problems and sometimes seizures progressed but affected mice could survive up to 5 months. The average survival was 122 ± 17 (SD) days (n = 20) (Fig. 3B).
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Pathology
The external aspects of the brains of saposin A–/– mice appeared grossly normal. Nerves were firm and abnormally thick, a feature also of GLD (Krabbe disease) in humans and other animals due to genetic GALC deficiency (Fig. 4A). Hind leg muscles showed severe atrophy, being replaced with fat in the terminal stage. Neuropathology can be summarized as a milder form of GLD as seen in human patients and other animal models, such as the twitcher mouse (13–15), due to genetic GALC deficiency. Progressive demyelination with infiltration of macrophages containing periodic acid Schiff (PAS)-positive materials was observed before 1 month, well in advance of detectable clinical symptoms, in the white matter of brain stem fiber tracts, cerebellum and spinal cord. The infiltration of macrophages became more conspicuous by 2 months. They had morphological characteristics of ‘globoid cells’ in GLD. They were large and multinucleated, clustered around blood vessels, PAS-positive and contained needle-shaped inclusions (Fig. 4B–D). Such inclusions were also seen within Schwann cells. Macrophages were widespread in the entire white matter including corpus callosum, internal capsule, pencil fibers in the striatum and brain stem fiber tracts. Notably, many macrophages with myelin droplets were present in the exit zone of the 5th cranial nerve. Peripheral nervous system (PNS) involvement was more extensive even compared with the twitcher mouse, which generally has a more severe disease (Fig. 4E and F). In the terminal stage, at
4–5 months, the spinal cord became grossly enlarged and showed severe demyelination with numerous multinucleated macrophages (globoid-like cells), especially in the gracile tract. The anterior spinal root and the root exit zone were also severely affected. The neuronal inclusions that appeared identical to those seen in long surviving twitcher mice (13) and also in later stages of twitcher mice with additional complete deficiency of GalCer synthase (16) were frequently seen in the reticular formation, anterior horn, hippocampal CA3 region and in the cerebral cortex. In the testis, the number of spermatocytes appeared within the normal range even in the 4-month-old mice (data not shown).
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Biochemistry
Sphingolipids of brain, kidney and testis were examined by thin-layer chromatography at 2, 4 and 5 months and compared with those of 45-day-old twitcher mice and 30-day-old total saposin-deficient mice. In the brain, saposin A–/– mice showed a slight increase in GalCer and monogalactosyl diglyceride (MGD) (Fig. 5A). The possible increase in brain GalCer is very minor and is being studied further with more precise, quantitative methods. Saposin A–/– mice showed a significantly higher level of GalCer in the kidney (Fig. 5B) and of the seminolipid precursor (1-alkyl, 2-acyl, galactosylglycerol) in the testis (Fig. 5C) compared with age-matched wild-type mice. However, even at 4 months, accumulation of GalCer in the kidney in saposin A–/– mice was less than that in 45-day-old twitcher mice (Fig. 5B). MGD and the seminolipid precursor are also substrates of GALC and seminolipid is essential in normal spermatogenesis (17).
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Psychosine (galactosylsphingosine) as a cytotoxic metabolite is considered to be critical in the pathogenesis of GLD (18,19). The brain psychosine level in the saposin A–/– mice was approximately twice the normal level; 57 ± 8 pmol/mg protein at 2 months (n = 7) and 63 ± 6 pmol/mg protein at 4 months (n = 7), in contrast to 30 ± 3 at 2 months (n = 3) and 34 ± 4 pmol/mg protein at 4 months (n = 3) in wild-type littermates. The difference between the saposin A–/– and wild-type mice is statistically highly significant (P < 0.0001). However, the accumulation was much milder compared with the twitcher mouse at 40 days, which is its terminal stage (233 ± 14 pmol/mg protein, n = 4). It is noteworthy that the brain psychosine level did not increase after 2 months. For comparison, we also measured brain psychosine in total saposin-deficient mice at their terminal stage of 40 days. The brain psychosine level in the total saposin-deficient mouse at 40 days was 17 ± 1.2 pmol/mg protein (n = 4) whereas in wild-type littermates at 40 days, analyzed at the same time, it was 34 ± 4 pmol/mg (n = 4). The difference was statistically highly significant (P = 0.0003). Thus, in contrast to either the saposin A–/– mouse or the twitcher mouse, brain psychosine in the total saposin-deficient mouse is lower than normal.
GALC activities were determined on brain homogenates. GALC activities in the brain of saposin A–/– mice were 0.74 ± 0.15 nmol/h/mg protein (n = 3), which was half the level in the brain of wild-type mice (1.41 ± 0.23 nmol/h/mg protein, n = 3, P = 0.013).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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The targeting construct had two features in addition to the standard inclusion of Neo gene and thymidine kinase (TK) gene for double selection of correctly targeted ES cells; presence of the loxP sequences flanking the Neo gene within an intron for later removal of Neo, and a newly generated DraI site for convenient genotyping. Even though the Neo gene was within an intron, we removed it because the presence of a foreign sequence of the size of the Neo gene would have been likely to result in abnormal splicing and an unstable message. We had considered application of Cre recombinase in vivo using Cre transgenic mice but eventually chose to remove Neo gene at the stage of targeted ES cells. Application of Cre recombinase using transient transfection of the Cre expression plasmid removed the Neo gene efficiently from ES cells. The single loxP sequence left within the intron appeared to have no ill effect in the splicing, judged from the size and stability of prosaposin mRNA.
All of the clinical, pathological and biochemical results observed in saposin A–/– mice not only confirmed earlier suggestive data that saposin A might be a GALC activator (7,8), but more importantly also established that it is in fact indispensable for normal in vivo catabolism of GalCer. However, the metabolic block of GalCer degradation due to saposin A deficiency appears less than complete, because the clinical, pathological and biochemical phenotype was generally much milder, with a conspicuous exception of the severe PNS involvement, than that of twitcher mice, which are totally deficient in GALC (20). We cannot exclude the possibility that other saposins, particularly saposin C, might contribute to GALC-activating function in the absence of saposin A, although they cannot completely compensate for the absence of saposin A (8,21). Since the only genetic lesion in the saposin A–/– mice is in saposin A, we had anticipated normal GALC activity in the conventional in vitro enzyme assay system using detergents. However, the activity was approximately half the normal level. The total saposin-deficient mouse also showed approximately half the normal level of GALC activity (6). Thus, our observations bring up an intriguing suggestion that saposin A may function as a stabilizer of GALC in addition to being its activator. Another point worth noting is the normal male fertility and the relatively pathology-free testis. Prosaposin gene also encodes the Sertoli cell major sulfated glycoprotein (22), which is considered important in normal function of male reproductive organs. The mouse mutant with total saposin deficiency indeed exhibits pathology in male reproductive organs (23). Despite the mutation near the N-terminus, the C106F mutation in the saposin A domain did not appear to affect normal function of male testis. In addition, prosaposin is reported to have functions of its own as a neurotrophic factor (24). Although prosaposin in saposin A–/– mice has an abnormal primary sequence, we did not find abnormalities indicative of loss of the neurotrophic function of prosaposin. More detailed analysis will be necessary to clarify this question definitively. In this regard, it should also be pointed out that prosaposin itself is abnormal in all human patients with mutations in either the saposin B or C domain. Although it has been proposed that a short peptide segment in domain C is responsible for the neurotrophic function (25), it remains to be demonstrated either that the likely abnormalities in the secondary and tertiary structures of these mutant prosaposin proteins do not affect the neurotrophic function of the peptide as long as the peptide sequence itself remains intact, or that some of these patients in fact have abnormalities in their brain development or function as the consequence of loss of the neurotrophic function of prosaposin.
Psychosine (galactosylsphingosine) is one of the substrates of GALC. It is a highly cytotoxic compound (26–30). It induces apoptosis in cultured cells as potently as C6-ceramide (31). At present, psychosine is known to be generated only by galactosylation of sphingosine. Enzymatic reaction between psychosine and GalCer in either direction has never been convincingly demonstrated in mammalian species. Psychosine may therefore be a dead-end metabolic product that is degraded rapidly in normal tissues. It is detectable in normal brain only at 15–30 pmol/mg protein, a level barely adequate for reliable determination by the present sensitive analytical technology. However, psychosine accumulates in human patients with Krabbe disease as well as in GLD, which occur in several mammalian species because of the underlying defect in the catabolism of GALC substrates (32). Experimental evidence has been accumulating in support of a hypothesis that psychosine may be the metabolite primarily responsible for the pathogenesis of GLD in the nervous system (18,19). It was therefore of interest to find that psychosine in the brain of saposin A–/– mice was only 2–3 times the normal level, in contrast to the 10- to 20-fold increase in the twitcher mouse brain. However, it is not possible to draw a logical conclusion that the significant but low accumulation of psychosine is causally related to the mild clinical, pathological and biochemical phenotype in the saposin A–/– mice.
A few points of uncertainty need to be pointed out which should be subjects for future studies. Firstly, the definitive proof that our mouse has specific saposin A deficiency with other three saposins completely intact is not yet on hand because of the limitation in the present technology. Ideally, it should be demonstrated that only saposin A protein is absent and the other three are normally present by immunochemical means. However, no specific individual anti-mouse saposin antibodies are available at present. We have attempted collaborations with two laboratories but the existing anti-mouse saposin antibodies available in one laboratory were unsuitable, and attempts at taking advantage of possible cross-reactivity with anti-human saposin antibodies available in the second laboratory were also unsuccessful. However, we feel comfortable to present the mouse as saposin A–/– based on the following several pieces of circumstantial evidence. Equivalent disease-causing mutations that replace one of the strictly conserved cysteines in the saposin B and C domains with phenylalanine or serine are known among human patients. In these patients, it has been shown that the initial translation product is normally processed and only the specific mutated saposin is absent while others are present and functionally intact (1). This was precisely the reason we introduced this specific mutation into the saposin A domain. The saposin A–/– mutant allele does generate a normal amount of stable mRNA (Fig. 2C). The substrate specificities of saposin B and C are well established through direct studies and also through human patients with specific genetic deficiencies. Saposin D deficiency is expected to result in abnormal ceramide degradation. Our mouse does not show any of the clinical, pathological or biochemical phenotypes we might expect if it were also deficient in any of the other three saposins.
The second uncertainty is the unexplained discrepancy between the total saposin-deficient mouse and the saposin A–/– mouse. The total saposin-deficient mouse exhibits an extremely severe, rapidly progressive disease affecting multiple organs and tissues. Naturally saposin A is deficient as well as the other three saposins. Nevertheless, no GLD-like pathology could be detected by a careful light and electron microscopic scrutiny (33). Initially, we thought the reason was simply because the total saposin-deficient mice do not live long enough to develop the characteristic late-onset GLD due to saposin A deficiency. However, we believe this explanation is untenable because more recent pathological studies of the saposin A–/– mice clearly indicated that the GLD pathology can be detected with relative ease already at 30 days. Many total saposin-deficient mice survive up to 40 days. More recent brain psychosine data in the total saposin-deficient mice showing only half the normal level further indicate that there must be reasons other than just disease duration or age of the animal to explain why the total saposin-deficient mouse does not show any of the phenotypic characteristics of the saposin A–/– mouse. At present, we can only conclude that the outcome of multiple saposin deficiency is not merely additive of individual saposin deficiency.
Finally, we have observed that a small but significant percentage of saposin A–/– male mice develop conspicuous hepatomegaly. The liver is approximately twice the normal size. Neurologically these mice are indistinguishable from other saposin A–/– mice. This phenotype has been observed so far in only 20% of affected males (10/49 saposin A–/– males compared with 0/47 saposin A–/– females, and also never among saposin A+/– or wild-type littermates, all older than 50 days). We are unable to offer a genetic explanation for this phenomenon. We still need more phenomenological observations and detailed pathological and biochemical studies of this apparent variant expression in order to eventually clarify this puzzling finding.
Despite the few remaining questions for future studies pointed out immediately above, the results presented here clearly demonstrate that genetic saposin A deficiency causes a late-onset, chronic form of GLD in the mouse. The clinical, biochemical and pathological features are qualitatively identical to, but milder than, those seen in the twitcher mouse. These results not only establish that saposin A is essential for in vivo degradation of GalCer but also anticipate genetic saposin A deficiency among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency. The saposin A–/– mouse can be useful for many experimental manipulations because defined genotypes can be generated reliably and efficiently, since both affected males and females are fertile.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Construction of saposin A targeting vector and generation of the saposin A mutant mice
Our cloning of mouse saposin gene from a mouse 129Sv genomic library has been described previously (6). To construct the targeting vector, two separate gene fragments were subcloned into pBluescript KS(+) (Stratagene); a 2.3 kb SalI/HindIII fragment containing exons 2 and 3 (subclone 1) and a 4.3 kb HindIII/EcoRI fragment containing exons 4 and 7 (subclone 2). Subclone 2 was used to introduce the Cys
Phe substitution at amino acid position 106 in exon 4 by using the four-oligonucleotide PCR mutagenesis method. The mismatch oligonucleotide sets were: primer 1, 5'-GTGGTTAGGCCGAGGTTGACCGCG-3' (located outside the unique BsmBI site); primer 2, 5'-CAACCACCTCTTTAAACGAGGCCGACA-3'; primer 3, 5'-CTGTCGGCCTCGTTTAAAGAGGTGGTT-3'; primer 4, 5'-GCCTTTCAGACTCTACAGAACTAGCTCTCTGGTT-3' (located outside the unique AccIII site). Primers 2 and 3 overlapped each other and contained the mutation (underlined). This mutation leads to the substitution of the 4th Cys and destruction of one of the disulfide bonds in saposin A, and simultaneously introduces a unique DraI recognition site for convenient genotyping. The mutant PCR products were digested with BsmBI and AccIII and replaced with the homologous fragment in subclone 2. The targeting construct was generated in the vector OSdupdel, which contains MC1-neomycin resistance gene (Neo) flanked by two loxP sites and TK under control of 3' phosphoglycerate kinase (PGK) (34) as follows. The homologous 1.3 kb BsaBI/HindIII fragment from subclone 1 was ligated to the KpnI/BamHI site of the vector downstream to the Neo selection cassette flanked by two loxP sites to form the 5' region of the homology. The homologous 4.3 kb HindIII/EcoRI fragment carrying the introduced C106F mutation was ligated to the XhoI/SfiI sites between the MC1-Neo and the TK-PKG genes to form the 3' homology region. The long and short homologous arms of saposin gene fragments were divided within an intron. The transcription orientations of the saposin gene and the Neo gene were opposite (Fig. 1). The targeting vector was linearized by NotI and electroporated into 2 x 107 ES cells derived from 129/SvEv strain, and stably transfected ES cell clones were isolated after double selection with G418 and Ganciclovir. Homologously recombined ES cell clones were identified by PCR using the primers from the 3' end of the Neo gene, 5'-CTTCTATCGCCTTCTTGACGAG-3', and just outside the short arm of the targeting vector, 5'-CTGATACCTGCCAGAGTTTGGT-3', indicated by arrows in Figure 1. A 1615 bp PCR product was generated from the recombinant ES cell clones and those were confirmed by Southern blot analysis using the combination of BamHI and DraI digestion with the 944 bp SnaBI/AviII fragment (3' probe), indicated in Figure 1 as the probe. Correctly targeted clones were transiently transfected with 20 µg of a CMV promoter-Cre expression plasmid, developed in the UNC Animal Models Core Facility, to remove the Neo cassette, and its successful removal was confirmed by PCR and Southern blot analysis. Positive mutant clones were used to produce chimeric animals by microinjection into C57BL/6J blastocysts. Male chimeras were then mated with female C57BL/6J mice for germ line transmission. Heterozygous F1 mice identified by PCR and Southern blot analysis of tail DNA were intercrossed to generate saposin A–/– mice.
Southern blot genotyping analysis
We used 944 bp SnaBI/AviII DNA fragments containing exons 9–10 located outside of the targeting vector on the 3' side as a probe to identify correctly targeted ES cell clones before and after Cre-treatment, and to confirm subsequent mutant mice. Probe was labeled with digoxigenin-dUTP (DIG-High Prime, Roche Diagnostics) according to the manufacture’s protocol. Briefly, 10 µg of genomic DNA was digested with BamHI and/or DraI and subjected to 0.8% agarose gel electrophoresis, which was then probed with DIG-labeled 3' probe. The hybridized probes were immunodetected with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (anti-DIG-AP) and then visualized with the chemiluminescent substrate CSPD. The hybridized membrane was exposed to X-ray film for 30 min at room temperature. The presence of a BamHI site and the DraI site introduced by the targeting vector leads to a change of fragment length from 14 kb in the wild-type allele to 7.8 kb in the recombinant allele. Single digestion with BamHI was also used to detect the correct Neo excision from the mutant allele after the Cre-treatment. Consequently, a 14 kb BamHI fragment from the wild-type allele, a 9.7 kb fragment from Neo+ targeted allele and a 8.5 kb fragment from correctly Neo-excised targeted allele were detected, respectively (Fig. 2A and B).
PCR genotyping analysis
To facilitate genotype analysis of the large number of saposin A–/– mice, we designed two oligonucleotide primers to distinguish the mutant allele from the wild-type allele by PCR using genomic DNA extracted from the tip of the clipped tail. The oligonucleotide primers were designed to flank the retained one loxP site in intron 3 of the mouse prosaposin gene (sense, 5'-GTCTGGCTTTCCAGGTCATACATA-3' and antisense, 5'-CACATGTAGGCAGAACACTCGTACAC-3'), indicated by arrows (Fig. 1). The PCR product of the wild-type allele was a 95 bp fragment, whereas the PCR product of the mutant allele was a 216 bp fragment (Fig. 2B).
Semiquantitative RT–PCR
Whole brain tissues from saposin A–/–, saposin A+/–, wild-type and total saposin-deficient mice were collected. The mice were killed by decapitation and the brains were rapidly dissected and processed for RNA extraction. Total RNA was isolated from each whole brain using TRI reagent (Sigma) following the manufacturer’s protocol. Total RNA (2.5 µg) was reverse-transcribed using the SuperScript first strand cDNA synthesis system (Gibco BRL) and oligo (dT)12–18 primers. The cDNA was then amplified using specific primers for mouse saposin and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The primers used were: sense, 5'-CTGTCGGCCTCGTGCAAGGAGGTGGTT-3'; antisense, 5'-GGCAGTCTCCATGTTCTGACAC-3' for mouse prosaposin [1323 bp (exons 4–14)]; and sense, 5'-CCATGGAGAAGGCCGGGG-3'; antisense, 5'-CAAAGTTGTCATGGATGACC-3' for mouse GAPDH (35). The optimized numbers of PCR cycles allowing the signal to be in the linear portion of the amplification curve were 20 cycles for prosaposin and 17 cycles for GAPDH. A negative control lacking template cDNA was included in each RT–PCR. For quantitative analysis, the stained gel was scanned with an image scanner (ScanJet ADF, Hewlett-Packard) and the band densities were digitized using the automated digitizing software (UN-SCAN-IT gel version 5.1, Silk Scientific). The values obtained for prosaposin bands were normalized for the GAPDH bands (Fig. 2C).
Animal care and clinical studies
All animal protocols used in these studies were approved by the Internal Review Board of our university. Mice were maintained with access to water ad libitum. 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 or other extraneous interventions. For biochemical and pathological evaluation, some mice were killed at 1, 2, 4 and 5 months.
Enzymatic assays
GALC activities were assayed on the brains of 2 to 4-month-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 (36). Activities of GALC were determined with tritium-labeled GalCer as substrate (37).
Histopathology
The mice were anesthetized with ether and perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, and immersed in the same fixative at 4°C overnight. Then tissues were processed for paraffin and plastic embedding, sectioned and analyzed by light and electron microscopy according to the standard procedure of our laboratory.
Lipid extraction from tissues
Tissues were homogenized with water at 20% of concentration by weight in an all-glass Potter–Elvehjem homogenizer. Initial extraction with chloroform-methanol was done as described previously (6). Brain, kidney and testis lipids were fractionated to neutral and acidic fractions using the reverse phase column essentially according to Kyrklund (38) (Varian Bond Elute, C-18; 3 ml/500 mg). This procedure gave the advantage of clean separation of all gangliosides and sulfatide into the acidic fraction, simple and rapid desalting and no need for saponification of the acidic fraction. Aliquots of the brain neutral lipid fraction were subjected to the mercuric chloride-saponification procedure in order to remove essentially all glycerophospholipids (39).
Quantitation of psychosine
Brain psychosine (galactosylsphingosine) was determined by the HPLC procedure (40,41), as modified by us more recently (42), except that the mobile phase was methanol-5 mM sodium phosphate buffer, pH 7.0 (89:11) with 50 mg/l sodium octylsulfate as a ion-pairing agent (16). The peaks of phytosphingosine, psychosine (galactosylsphingosine), sphingosine, sphinganine and eicosasphinganine (internal standard) were eluted in this 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. However, our experience with GalCer synthase-deficient mice indicates that mouse brain contains no detectable amount of glucosylsphingosine, with the sole exception of glucosylceramidase-deficient (Gaucher) mouse (16). 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 the unpaired Student’s t-test.
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ACKNOWLEDGMENTS |
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The authors thank Dr Nobuyo Maeda for providing us with the vector (OSdupdel), Dr Randy J. Thresher in the University of North Carolina Animal Models Core Facility for helping with ES cell handling, Ms Elise Cash and Clarita Langaman for the tissue preparation and Mr Joe Langaman for cell culture and other technical assistance. This work was supported in part by research grants, RO1-NS24289 and a Mental Retardation Research Center Core Grant, P30-HD03110, from the USPHS, and a research grant 83A from the Mizutani Foundation to Kunihiko Suzuki; 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: Nishi-Niigata Central Hospital, 1-14-1 Masago, Niigata 950-2085, 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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1 Sandhoff, K., Kolter, T. and Harzer, K. (2001) 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. 3371–3388.
2 Christomanou, H., Aignesberger, A. and Link, R.P. (1986) Immunochemical characterization of two activator proteins stimulating enzymic sphingomyelin degradation in vitro: Absence of one of them in a human Gaucher disease variant. Biol. Chem. Hoppe Seyler, 367, 879–890.[Web of Science][Medline]
3 Stevens, R.L., Fluharty, A.L., Kihara, H., Kaback, M.M., Shapiro, L.J., Marsh, B., Sandhoff, K. and Fischer, G. (1981) Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy. Am. J. Hum. Genet., 33, 900–906.[Web of Science][Medline]
4 Schnabel, D., Schröder, M., Fürst, W., Klein, A., Hurwitz, R., Zenk, T., Weber, J., Harzer, K., Paton, B.C., Poulos, A. et al. (1992) Simultaneous deficiency of sphingolipid activator proteins 1 and 2 is caused by a mutation in the initiation codon of their common gene. J. Biol. Chem., 267, 3312–3315.
5 Hùlková, H., Cervenková, M., Ledvinová, J., Tocháèková, M., Hrebíèek ,M., Poupetová, H., Befekadu, A., Berná, L., Paton, B.C., Harzer, K. et al. (2001) A novel mutation in the coding region of the prosaposin gene leads to a complete deficiency of prosaposin and saposins, and is associated with a complex sphingolipidosis dominated by lactosylceramide accumulation. Hum. Mol. Genet., 10, 927–940.
6 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.
7 Morimoto, S., Martin, B.M., Yamamoto, Y., Kretz, K.A., O’Brien, J.S. and Kishimoto, Y. (1989) Saposin-A—2nd cerebrosidase activator protein. Proc. Natl Acad. Sci. USA, 86, 3389–3393.
8 Harzer, K., Paton, B.C., Christomanou, H., Chatelut, M., Levade, T., Hiraiwa, M. and O’Brien, J.S. (1997) Saposins (sap) A and C activate the degradation of galactosylceramide in living cells. FEBS Lett., 417, 270–274.[Web of Science][Medline]
9 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]
10 Azuma, N., O’Brien, J.S., Moser, H.W. and Kishimoto, Y. (1994) Stimulation of ceramidase by saposin D. Trans. Am. Soc. Neurochem., 25, 254.
11 Sauer, B. and Herderson, N. (1989) Cre-stimulated recombination at loxP-containing DNA sequence placed into the mammalian genome. Nucleic Acids Res., 17, 147–161.
12 Vaccaro, A.M., Salvioli, R., Barca, A., Tatti, M., Ciaffoni, F., Maras, B., Siciliano, R., Zappacosta, F., Amoresano, A. and Pucci, P. (1995) Structural analysis of saposin C and B. Complete localization of disulfide bridges. J. Biol. Chem., 270, 9953–9960.
13 Duchen, L.W., Eicher, E.M., Jacobs, J.M., Scaravilli, F. and Teixeira, F. (1980) Hereditary leukodystrophy in the mouse—the new mutant twitcher. Brain, 103, 695–710.
14 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]
15 Suzuki, K. and Suzuki, K. (1995) The twitcher mouse: A model for Krabbe disease and for experimental therapies. Brain Pathol., 5, 249–258.[Web of Science][Medline]
16 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]
17 Fujimoto, H., Tadano-Aritomi, K., Tokumasu, A., Ito, K., Hikita, T., Suzuki, K. and Ishizuka, I. (2000) Requirement of seminolipid in spermatogenesis revealed by UDP-galactose:ceramide galactosyltransferase-deficient mice. J. Biol. Chem., 275, 22623–22626.
18 Miyatake, T. and Suzuki, K. (1972) Globoid cell leukodystrophy: Additional deficiency of psychosine galactosidase. Biochem. Biophys. Res. Commun., 48, 538–543.[Web of Science]
19 Suzuki, K. (1998) Twenty five years of the psychosine hypothesis: A personal perspective of its history and present status. Neurochem. Res., 23, 251–259.[Web of Science][Medline]
20 Sakai, N., Inui, K., Tatsumi, N., Fukushima, H., Nishigaki, T., Taniike, M., Nishimoto, J., Tsukamoto, H., Yanagihara, I., Ozone, K. et al. (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]
21 Wenger, D.A., Sattler, M. and Roth, S. (1982) A protein activator of galactosylceramide ß-galactosidase. Biochim. Biophys. Acta, 712, 639–649.[Medline]
22 Collard, M.W., Sylvester, S.R., Tsuruta, J.K. and Griswold, M.D. (1988) Biosynthesis and molecular cloning of sulfated glycoprotein I secreted by rat Sertoli cells: Sequence similarity with the 70-kilodalton precursor to sulfatide GM1 activator. Biochemistry, 27, 4557–4564.[Medline]
23 Morales, C.R., Zhao, Q., El-Alfy, M. and Suzuki, K. (2000) Targeted disruption of the mouse prosaposin gene affects the development of the prostate gland and other male reproductive organs. J. Androl., 21, 765–775.[Abstract]
24 O’Brien, J.S., Carson, G.S., Seo, H.-C., Hiraiwa, M. and Kishimoto, Y. (1994) Identification of prosaposin as a neurotrophic factor. Proc. Natl Acad. Sci. USA, 91, 9593–9596.
25 O’Brien, J.S., Carson, G.S., Seo, H.-C., Hiraiwa, M., Weiler, S., Tomich, J.M., Barranger, J.A., Kahn, M., Azuma, N. and Kishimoto, Y. (1995) Identification of the neurotrophic factor sequence of prosaposin. FASEB J., 9, 681–685.[Abstract]
26 Taketomi, T. and Nishimura, K. (1964) Physiological activity of psychosine. Jpn. J. Exp. Med., 34, 255–265.[Medline]
27 Sugama, S., Kim, S.U., Ida, H. and Eto, Y. (1990) Psychosine cytotoxicity in rat neural cell cultures and protection by phorbol ester and dimethyl sulfoxide. Pediatr. Res., 28, 473–476.[Web of Science][Medline]
28 Igisu, H. and Nakamura, M. (1986) Inhibition of cytochrome c oxidase by psychosine (galactosylsphingosine). Biochem. Biophys. Res. Commun., 137, 323–327.[Web of Science][Medline]
29 Igisu, H., Matsuoka, M. and Hamasaki, N. (1990) Binding of galactosylsphingosine (psychosine) by albumin. Lipids, 25, 65–68.[Web of Science][Medline]
30 Hannun, Y.A. and Bell, R.M. (1987) Lysosphingolipids inhibit protein kinase C: Implications for the sphingolipidoses. Science, 235, 670–674.
31 Tohyama, J., Matsuda, J. and Suzuki, K. (2001) Psychosine is as potent an inducer of cell death as C6-ceramide in cultured fibroblasts and in MOCH cells. Neurochem. Res., in press.
32 Wenger, D.A., Suzuki, K., Suzuki, Y. and Suzuki, Y. (2001) 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. 3669–2694.
33 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. (Berl.), 96, 29–40.[Medline]
34 Wang, Y., Spatz, M.K., Kannan, K., Hayk, H., Avivi, A., Gorivodsky, M., Pines, M., Yayon, A., Lonai, P. and Givol, D. (1999) A mouse model for achondroplasia produced by targeting fibroblast growth factor receptor 3. Proc. Natl Acad. Sci. USA, 96, 4455–4460.
35 Wu, Y.P., Matsuda, J., Kubota, A., Suzuki, K. and Suzuki, K. (2000) Infiltration of hematogenous lineage cells into the demyelinating central nervous system of twitcher mice. J. Neuropathol. Exp. Neurol., 59, 628–639.[Web of Science][Medline]
36 Hartree, E.F. (1972) Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal. Biochem., 48, 122–127.
37 Vanier, M.T., Svennerholm, L., Månsson, J.-E., Håkansson, G., Boue, A. and Lindsten, J. (1981) Prenatal diagnosis of Krabbe disease. Clin. Genet., 20, 79–89.[Web of Science][Medline]
38 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]
39 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.
40 Merrill, A.H.,Jr, 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]
41 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]
42 Matsumoto, A., Vanier, M.T., Oya, Y., Kelly, D., Popko, B., Wenger, D.A., Suzuki, K. and Suzuki, K. (1997) Transgenic introduction of human galactosylceramidase into twitcher mouse: Significant phenotype improvement with a minimal expression. Dev. Brain Dysfunc., 10, 142–154.
43 Tohyama, J., Vanier, M.T., Suzuki, K., Ezoe, T., Matsuda, J. and Suzuki, K. (2000) Paradoxical influence of acid ß-galactosidase gene dosage on phenotype of the twitcher mouse (genetic galactosylceramidase deficiency). Hum. Mol. Genet., 9, 1699–1707.
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