A Mutation in the Saposin a Domain of the Sphingolipid Activator Protein

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

Junko Matsuda1,MarieT.Vanier4,YukoSaito3,JunTohyama1,+, Kinuko Suzuki1,3 and Kunihiko Suzuki1,2,§

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

Sphingolipid activator proteins (saposins A, B, C and INTRODUCTION D) are small homologous glycoproteins derived from Sphingolipid activator proteins (saposins A, B, C and D) are small a common precursor protein (prosaposin) encoded heat-stable glycoproteins required for in vivo degradation of some by a single gene. They are required for in vivo degrad- sphingolipids with short carbohydrate chains (1). They are ation of sphingolipids with short carbohydrate derived from a common precursor protein (prosaposin), which chains. Six cysteines and one glycosylation site are is encoded by a single gene, and proteolytically processed to strictly conserved in all four saposins. Total deficiency saposins A, B, C and D. These four saposins are all homologous to each other, having six conserved cysteines and one common of all saposins and specific deficiency of saposin B glycosylation site. Inspite of these structural similarities, their or C are known among human patients. A mouse activator functions are specific, with some overlaps, for model of total saposin deficiency closely mimics the individual sphingolipid hydrolases. Human patients with human disease. However, no specific saposin A or D mutations in the saposin B and C domains are known and they deficiency is known. We introduced an amino acid show phenotypes of metachromatic leukodystrophy and substitution (C106F) into the saposin A domain by Gaucher disease, indicating that their primary in vivo the Cre/loxP system which eliminated one of the substrates are sulfatide and glucosylceramide, respectively (2,3). Two mutations are known in humans which result in three conserved disulfide bonds. Saposin A–/– mice complete inactivation of all four saposins and prosaposin (4,5). developed slowly progressive hind leg paralysis with In addition, we previously generated a mouse model of total clinical onset at ∼2.5 months and survival up to 5 saposin deficiency with the gene-targeting technology that months. Tremors and shaking, prominent in other closely mimics the human disease (6). Total saposin myelin mutants, were not obvious until the terminal deficiency, both in humans and mice, is a devastating disease stage. Pathology and analytical biochemistry were with a complex phenotype involving multiple organs and qualitatively identical to, but generally much milder multiple sphingolipids, indicating the essential roles of these in vivo than, that seen in the typical infantile globoid cell saposins . There are reports suggestive of saposin A being a galactosylceramidase (GALC) activator (7,8), and leukodystrophy (GLD) in man (Krabbe disease) and saposin D being a ceramidase activator (9,10). However, in several other mammalian species, due to genetic absence of specific genetic deficiency of either saposin A or D deficiency of lysosomal galactosylceramidase (GALC) leaves unanswered the question of whether either saposin A or (EC 3.2.1.46). Thus, saposin A is indispensable for D is indispensable for normal cellular function. We introduced in vivo degradation of galactosylceramide by GALC. an amino acid substitution (C106F) into the saposin A domain It should now be recognized that, in addition to GALC by the Cre/loxP system (11) which eliminated one of the three deficiency, genetic saposin A deficiency could also disulfide bonds. The maintenance of the three strictly conserved disulfide bridges is considered essential for the cause chronic GLD. Genetic saposin A deficiency functional properties of saposins (12). In humans, an equivalent might be anticipated among human patients with mutation in the 4th cysteine to phenylalanine in saposin C undiagnosed late-onset chronic leukodystrophy causes specific saposin C deficiency, and a mutation of the 5th without GALC deficiency. cysteine to serine in saposin B causes specific saposin B

+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: [email protected] 1192 Human Molecular Genetics, 2001, Vol. 10, No. 11

Figure 1. Schematic diagrams and partial restriction maps of the mouse saposin locus (wild-type), saposin A targeting vector (containing exons 2–7) after mutating Cys at 106 to phenylalanine (Phe), and mutant saposin allele. The targeting vector was constructed to introduce a mutation in exon 4 that changed the 4thofthe six strictly conserved cysteine (Cys) residues to Phe, resulting in the destruction of one of the disulfide bridges in saposin A. The vector sequence also introduced anewDraI site. The amino acid sequence in the construct was otherwise unchanged. In addition to the mutation, a neomycin-resistance gene (Neo) flanked by the loxP sequences was present in the intronic sequence of the targeting vector. These allowed initial selection of targeted ES cells by neomycin and facile identification of targeted ES cells by DraIsite.TheNeo gene and one loxP site were subsequently removed by Cre recombinase before the ES cells were introduced into blastocysts. The numbered boxes represent the saposin exons. The bold line represents the 3′-flanking probe used to identify the targeted allele. The open boxes indicate either the MC1-Neo or the PGK-TK gene. B, BamHI; H, HindIII; E, EcoRI. deficiency (1). In these human disorders of saposin B and C deficiency (Fig. 2C). Viable saposin A–/– mice were obtained deficiency, the other unaffected saposins are properly processed with the frequency expected from the Mendelian principle. and fully functional. The saposin A–/– mice developed a late- Saposin A–/– mice initially grew normally and were indistin- onset, chronic form of globoid cell leukodystrophy (GLD), guishable from their littermates. However, at ∼2.5 months, clearly indicating that saposin A is indispensable for GALC to careful observation could distinguish affected mice from litter- degrade galactosylceramide (GalCer) in vivo and that genetic mates by their subtle hind leg weakness and sluggish activities. saposin A deficiency might be anticipated among human Paralysis and atrophy of hind legs progressed slowly and the patients with undiagnosed late-onset chronic leukodystrophy, mice stopped gaining weight at ∼3 months (Fig. 3A). Some of without GALC deficiency. the saposin A–/– mice showed generalized seizures and hyper- activity at ∼3 months. Twitching, prominently seen in twitcher RESULTS mice and other myelin mutants, did not become obvious until the end stage. Both males and females were fertile, and females Gene targeting and clinical phenotype were able to raise their offspring normally at least twice. Ileus and neurogenic bladder, reflecting autonomic nervous system The targeting vector was designed to introduce a mutation in dysfunction, were commonly seen, as is also the case in human exon 4 that changed the 4th cysteine in saposin A to phenyl- adult-onset leukodystrophy. Gait disturbance, feeding problems alanine, resulting in destruction of one of the disulfide bridges and sometimes seizures progressed but affected mice could in saposin A (Fig. 1). Correctly targeted embryonic stem (ES) survive up to 5 months. The average survival was 122 ± 17 (SD) cells, before and after removal of the neomycin resistance gene days (n =20)(Fig.3B). (Neo) by Cre recombinase, and genotypes of resultant mice were confirmed by appropriate DNA analyses (Fig. 2A and B). Pathology The allele with the C106F mutation in the saposin A domain generated stable prosaposin mRNA of normal length, as The external aspects of the brains of saposin A–/– mice expected, in contrast to its absence in the total saposin appeared grossly normal. Nerves were firm and abnormally Human Molecular Genetics, 2001, Vol. 10, No. 11 1193

Figure 2. (A) Southern blot analysis to detect mutated ES cells and correct Neo excision after transient transfection of Cre expression plasmid. Genomic DNA was digested with BamHI and DraI and hybridized with the probe depicted in Figure 1. The wild-type allele gives a band of ∼14 kb. Excision of Neo resulted in reduction of the size of the recombinant allele from ∼9.7 to ∼8.5kbwithBamHI digestion. (B) Left; Southern blot analysis of tail DNA from wild-type (+/+), saposin A (+/–) and saposin A (–/–) F2 progeny. Genomic DNA was digested with BamHI and DraI and hybridized with the probe depicted in Figure 1. The ∼ ∼ expected sizes of the BamHI/DraI fragments are 14 and 7.8 kb in the wild-type and recombinant allele, respectively. PCR analysis of tail DNA from F2 progeny is also shown (right). The PCR product of the wild-type allele is 95 bp, whereas the PCR product of the mutant allele is 216 bp. (C) Semi-quantitative RT–PCR analysis of prosaposin mRNA expression. Normal level prosaposin mRNA expression in saposin A (+/–) and saposin A (–/–) F2 progeny mouse brain, in contrast to its absence in total saposin-deficient mouse brain. Semi-quantitative RT–PCR was performed as described in detail in the text.

thick, a feature also of GLD (Krabbe disease) in humans and (PAS)-positive materials was observed before 1 month, well in other animals due to genetic GALC deficiency (Fig. 4A). Hind advance of detectable clinical symptoms, in the white matter of leg muscles showed severe atrophy, being replaced with fat in brain stem fiber tracts, cerebellum and spinal cord. The infiltration the terminal stage. Neuropathology can be summarized as a of macrophages became more conspicuous by 2 months. They milder form of GLD as seen in human patients and other had morphological characteristics of ‘globoid cells’ in GLD. animal models, such as the twitcher mouse (13–15), due to They were large and multinucleated, clustered around blood genetic GALC deficiency. Progressive demyelination with vessels, PAS-positive and contained needle-shaped inclusions infiltration of macrophages containing periodic acid Schiff (Fig. 4B–D). Such inclusions were also seen within Schwann 1194 Human Molecular Genetics, 2001, Vol. 10, No. 11

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). 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)and34± 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 Figure 3. (A) Body weight of the saposin A–/– mice compared with that of wild-type total saposin-deficient mouse at 40 days was 17 ± 1.2 pmol/mg and twitcher mice. Each line represents the average of four or five representative protein (n = 4) whereas in wild-type littermates at 40 days, mice of the respective genotype. (B)LifespanofthesaposinA–/– mice (mean = analyzed at the same time, it was 34 ± 4pmol/mg(n =4).The ± ± 122 17 days) compared with that of twitcher mice (mean = 48 5days).The difference was statistically highly significant (P =0.0003). values for twitcher mice are from Tohyama et al. (43). Thus,incontrasttoeitherthesaposinA–/– mouse or the twitcher mouse, brain psychosine in the total saposin-deficient mouse is lower than normal. cells. Macrophages were widespread in the entire white matter GALC activities were determined on brain homogenates. including corpus callosum, internal capsule, pencil fibers in the GALC activities in the brain of saposin A–/– mice were 0.74 ± striatum and brain stem fiber tracts. Notably, many macro- 0.15 nmol/h/mg protein (n = 3), which was half the level in the phages with myelin droplets were present in the exit zone of brain of wild-type mice (1.41 ± 0.23 nmol/h/mg protein, n =3, the 5th cranial nerve. Peripheral nervous system (PNS) P =0.013). 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 DISCUSSION spinal cord became grossly enlarged and showed severe The targeting construct had two features in addition to the demyelination with numerous multinucleated macrophages standard inclusion of Neo gene and thymidine kinase (TK) (globoid-like cells), especially in the gracile tract. The anterior gene for double selection of correctly targeted ES cells; spinal root and the root exit zone were also severely affected. presence of the loxP sequences flanking the Neo gene within an The neuronal inclusions that appeared identical to those seen in intron for later removal of Neo, and a newly generated DraI long surviving twitcher mice (13) and also in later stages of site for convenient genotyping. Even though the Neo gene was twitcher mice with additional complete deficiency of GalCer within an intron, we removed it because the presence of a synthase (16) were frequently seen in the reticular formation, foreign sequence of the size of the Neo gene would have been anterior horn, hippocampal CA3 region and in the cerebral likely to result in abnormal splicing and an unstable message. cortex. In the testis, the number of spermatocytes appeared We had considered application of Cre recombinase in vivo within the normal range even in the 4-month-old mice (data not using Cre transgenic mice but eventually chose to remove Neo shown). gene at the stage of targeted ES cells. Application of Cre recombinase using transient transfection of the Cre expression Biochemistry plasmid removed the Neo gene efficiently from ES cells. The single loxP sequence left within the intron appeared to have no Sphingolipids of brain, kidney and testis were examined by ill effect in the splicing, judged from the size and stability of thin-layer chromatography at 2, 4 and 5 months and compared prosaposin mRNA. Human Molecular Genetics, 2001, Vol. 10, No. 11 1195

Figure 4. Pathology of the saposin A–/– mouse. (A) Sciatic nerve from a saposin A–/– mouse (right) compared with that from an age-matched wild-type mouse (left). All peripheral nerves of the saposin A–/– mouse are enormously thick. (B and C) Luxol-Fast Blue (LFB)-periodic acid Schiff (PAS) staining of the cerebellum from a saposin A–/– mouse at 4 months (B) and from a twitcher mouse at 35 days (C). The twitcher mouse has a much more extensive loss of myelin and more infiltration of the characteristic globoid cells at a much earlier age. (D) An electron micrograph of a ‘globoid cell’ which contains the characteristic needle-shaped inclusions (arrow). (E and F) LFB-PAS staining of the sciatic nerve from a saposin A–/– mouse at 50 days (E) and from a twitcher mouse at 25 days (F). Both mutants exhibited extensive myelin destruction and infiltration of the characteristic globoid cells. Although not shown, myelin droplets in the paranodal Schwann cell cytoplasm were also noted.

All of the clinical, pathological and biochemical results reproductive organs. The mouse mutant with total saposin observed in saposin A–/– mice not only confirmed earlier deficiency indeed exhibits pathology in male reproductive suggestive data that saposin A might be a GALC activator organs (23). Despite the mutation near the N-terminus, the (7,8), but more importantly also established that it is in fact C106F mutation in the saposin A domain did not appear to indispensable for normal in vivo catabolism of GalCer. affect normal function of male testis. In addition, prosaposin is However, the metabolic block of GalCer degradation due to reported to have functions of its own as a neurotrophic factor saposin A deficiency appears less than complete, because the (24). Although prosaposin in saposin A–/– mice has an clinical, pathological and biochemical phenotype was gener- abnormal primary sequence, we did not find abnormalities ally much milder, with a conspicuous exception of the severe indicative of loss of the neurotrophic function of prosaposin. PNS involvement, than that of twitcher mice, which are totally More detailed analysis will be necessary to clarify this deficient in GALC (20). We cannot exclude the possibility that question definitively. In this regard, it should also be pointed other saposins, particularly saposin C, might contribute to out that prosaposin itself is abnormal in all human patients with GALC-activating function in the absence of saposin A, mutations in either the saposin B or C domain. Although it has although they cannot completely compensate for the absence been proposed that a short peptide segment in domain C is of saposin A (8,21). Since the only genetic lesion in the saposin responsible for the neurotrophic function (25), it remains to be A–/– mice is in saposin A, we had anticipated normal GALC demonstrated either that the likely abnormalities in the activity in the conventional in vitro enzyme assay system using secondary and tertiary structures of these mutant prosaposin detergents. However, the activity was approximately half the proteins do not affect the neurotrophic function of the peptide normal level. The total saposin-deficient mouse also showed as long as the peptide sequence itself remains intact, or that approximately half the normal level of GALC activity (6). some of these patients in fact have abnormalities in their brain Thus, our observations bring up an intriguing suggestion that development or function as the consequence of loss of the saposin A may function as a stabilizer of GALC in addition to neurotrophic function of prosaposin. being its activator. Another point worth noting is the normal Psychosine (galactosylsphingosine) is one of the substrates male fertility and the relatively pathology-free testis. Prosaposin of GALC. It is a highly cytotoxic compound (26–30). It gene also encodes the Sertoli cell major sulfated glycoprotein induces apoptosis in cultured cells as potently as C6-ceramide (22), which is considered important in normal function of male (31). At present, psychosine is known to be generated only by 1196 Human Molecular Genetics, 2001, Vol. 10, No. 11

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 collab- orations 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 comfort- able to present the mouse as saposin A–/– basedonthe following several pieces of circumstantial evidence. Equiva- lent 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 Figure 5. Thin-layer chromatography of lipids from tissues of saposin A–/– mouse patients with specific genetic deficiencies. Saposin D deficiency at different ages. Solvent system: chloroform-methanol-water (65:25:4 by volume). is expected to result in abnormal ceramide degradation. Our Detection: orcinol spray. (A) Brain total lipid fraction. Each lane represents lipids mouse does not show any of the clinical, pathological or from 2 mg equivalent of wet tissue. Slight accumulation of GalCer is present in biochemical phenotypes we might expect if it were also –/– the saposin A mouse. Monogalactosyl diglyceride (MGD), another substrate deficient in any of the other three saposins. of GALC, is also slightly increased. (B) Kidney total lipid fraction. Each lane represents lipids from 5 mg equivalent of wet tissue. Significant accumulation The second uncertainty is the unexplained discrepancy of GalCer is obvious in the saposin A–/– mousekidneybutinalesserdegreethan between the total saposin-deficient mouse and the saposin A–/– seen in the kidney of twitcher mouse of a much younger age. (C) Testis total mouse. The total saposin-deficient mouse exhibits an lipid fraction. Each lane represents lipids from 5 mg equivalent of wet tissue. extremely severe, rapidly progressive disease affecting Accumulation of seminolipid precursor (1-alkyl,2-acyl,galactosylglycerol, GalEAG) is conspicuous in the saposin A–/– mouse testis. 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 galactosylation of sphingosine. Enzymatic reaction between simply because the total saposin-deficient mice do not live psychosine and GalCer in either direction has never been long enough to develop the characteristic late-onset GLD due convincingly demonstrated in mammalian species. Psychosine to saposin A deficiency. However, we believe this explanation Human Molecular Genetics, 2001, Vol. 10, No. 11 1197 is untenable because more recent pathological studies of the (underlined). This mutation leads to the substitution of the 4th saposin A–/– mice clearly indicated that the GLD pathology can Cys and destruction of one of the disulfide bonds in saposin A, be detected with relative ease already at 30 days. Many total and simultaneously introduces a unique DraI recognition site saposin-deficient mice survive up to 40 days. More recent for convenient genotyping. The mutant PCR products were brain psychosine data in the total saposin-deficient mice digested with BsmBI and AccIII and replaced with the homolo- showing only half the normal level further indicate that there gous fragment in subclone 2. The targeting construct was gen- must be reasons other than just disease duration or age of the erated in the vector OSdupdel, which contains MC1-neomycin animal to explain why the total saposin-deficient mouse does resistance gene (Neo)flankedbytwoloxP sites and TK under not show any of the phenotypic characteristics of the saposin control of 3′ phosphoglycerate kinase (PGK)(34)asfollows. A–/– mouse. At present, we can only conclude that the outcome The homologous 1.3 kb BsaBI/HindIII fragment from sub- of multiple saposin deficiency is not merely additive of clone 1 was ligated to the KpnI/BamHI site of the vector down- individual saposin deficiency. stream to the Neo selection cassette flanked by two loxPsites Finally, we have observed that a small but significant to form the 5′ region of the homology. The homologous 4.3 kb percentage of saposin A–/– male mice develop conspicuous HindIII/EcoRI fragment carrying the introduced C106F mutation hepatomegaly. The liver is approximately twice the normal was ligated to the XhoI/SfiI sites between the MC1-Neo and the size. Neurologically these mice are indistinguishable from TK-PKG genes to form the 3′ homology region. The long and other saposin A–/– mice. This phenotype has been observed so short homologous arms of saposin gene fragments were far in only ∼20% of affected males (10/49 saposin A–/– males divided within an intron. The transcription orientations of the compared with 0/47 saposin A–/– females, and also never saposin gene and the Neo gene were opposite (Fig. 1). The among saposin A+/– or wild-type littermates, all older than targeting vector was linearized by NotI and electroporated into 50 days). We are unable to offer a genetic explanation for this 2 × 107 ES cells derived from 129/SvEv strain, and stably phenomenon. We still need more phenomenological observations transfected ES cell clones were isolated after double selection and detailed pathological and biochemical studies of this with G418 and Ganciclovir. Homologously recombined ES apparent variant expression in order to eventually clarify this cell clones were identified by PCR using the primers from the puzzling finding. 3′ end of the Neo gene, 5′-CTTCTATCGCCTTCTTGAC- Despite the few remaining questions for future studies GAG-3′, and just outside the short arm of the targeting vector, pointed out immediately above, the results presented here 5′-CTGATACCTGCCAGAGTTTGGT-3′, indicated by arrows clearly demonstrate that genetic saposin A deficiency causes a in Figure 1. A 1615 bp PCR product was generated from the late-onset, chronic form of GLD in the mouse. The clinical, recombinant ES cell clones and those were confirmed by biochemical and pathological features are qualitatively identical Southern blot analysis using the combination of BamHI and to, but milder than, those seen in the twitcher mouse. These DraI digestion with the 944 bp SnaBI/AviII fragment (3′ results not only establish that saposin A is essential for in vivo probe), indicated in Figure 1 as the probe. Correctly targeted degradation of GalCer but also anticipate genetic saposin A clones were transiently transfected with 20 µgofaCMV deficiency among human patients with undiagnosed late-onset promoter-Cre expression plasmid, developed in the UNC chronic leukodystrophy without GALC deficiency. The Animal Models Core Facility, to remove the Neo cassette, and –/– saposin A mouse can be useful for many experimental its successful removal was confirmed by PCR and Southern manipulations because defined genotypes can be generated blot analysis. Positive mutant clones were used to produce reliably and efficiently, since both affected males and females chimeric animals by microinjection into C57BL/6J blastocysts. are fertile. Male chimeras were then mated with female C57BL/6J mice for germ line transmission. Heterozygous F1 mice identified by MATERIALSANDMETHODS PCR and Southern blot analysis of tail DNA were intercrossed to generate saposin A–/– mice. Construction of saposin A targeting vector and generation of the saposin A mutant mice Southern blot genotyping analysis Our cloning of mouse saposin gene from a mouse 129Sv We used 944 bp SnaBI/AviII DNA fragments containing exons genomic library has been described previously (6). To 9–10 located outside of the targeting vector on the 3′ side as a construct the targeting vector, two separate gene fragments probe to identify correctly targeted ES cell clones before and were subcloned into pBluescript KS(+) (Stratagene); a 2.3 kb after Cre-treatment, and to confirm subsequent mutant mice. SalI/HindIII fragment containing exons 2 and 3 (subclone 1) Probe was labeled with digoxigenin-dUTP (DIG-High Prime, and a 4.3 kb HindIII/EcoRI fragment containing exons 4 and 7 Roche Diagnostics) according to the manufacture’s protocol. (subclone 2). Subclone 2 was used to introduce the Cys→Phe Briefly, ∼10 µg of genomic DNA was digested with BamHI substitution at amino acid position 106 in exon 4 by using the and/or DraI and subjected to 0.8% agarose gel electrophoresis, four-oligonucleotide PCR mutagenesis method. The mismatch which was then probed with DIG-labeled 3′ probe. The hybrid- oligonucleotide sets were: primer 1, 5′-GTGGTTAGGCC- ized probes were immunodetected with anti-digoxigenin Fab GAGGTTGACCGCG-3′ (located outside the unique BsmBI site); fragments conjugated to alkaline phosphatase (anti-DIG-AP) primer 2, 5′-CAACCACCTCTTTAAACGAGGCCGACA-3′; and then visualized with the chemiluminescent substrate CSPD. primer 3, 5′-CTGTCGGCCTCGTTTAAAGAGGTGGTT-3′; The hybridized membrane was exposed to X-ray film for ∼30 min primer 4, 5′-GCCTTTCAGACTCTACAGAACTAGCTCTC- at room temperature. The presence of a BamHI site and the DraI TGGTT-3′ (located outside the unique AccIII site). Primers 2 site introduced by the targeting vector leads to a change of frag- and 3 overlapped each other and contained the mutation ment length from ∼14 kb in the wild-type allele to ∼7.8kbinthe 1198 Human Molecular Genetics, 2001, Vol. 10, No. 11 recombinant allele. Single digestion with BamHI was also used Enzymatic assays to detect the correct Neo excision from the mutant allele after GALC activities were assayed on the brains of 2 to 4-month- the Cre-treatment. Consequently, a ∼14 kb BamHI fragment old mice of each genotype. Freshly obtained brains were from the wild-type allele, a ∼9.7kbfragmentfromNeo+ homogenized with double distilled water (20%, w/v). The targeted allele and a ∼8.5 kb fragment from correctly Neo-excised protein content was determined by a modified Lowry method targeted allele were detected, respectively (Fig. 2A and B). (36). Activities of GALC were determined with tritium-labeled GalCer as substrate (37). PCR genotyping analysis To facilitate genotype analysis of the large number of saposin Histopathology –/– A mice, we designed two oligonucleotide primers to distin- The mice were anesthetized with ether and perfused with 4% guish the mutant allele from the wild-type allele by PCR using paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, genomic DNA extracted from the tip of the clipped tail. The andimmersedinthesamefixativeat4°C overnight. Then oligonucleotide primers were designed to flank the retained tissues were processed for paraffin and plastic embedding, one loxP site in intron 3 of the mouse prosaposin gene (sense, sectioned and analyzed by light and electron microscopy ′ ′ 5 -GTCTGGCTTTCCAGGTCATACATA-3 and antisense, according to the standard procedure of our laboratory. 5′-CACATGTAGGCAGAACACTCGTACAC-3′), indicated by arrows (Fig. 1). The PCR product of the wild-type allele Lipid extraction from tissues was a 95 bp fragment, whereas the PCR product of the mutant allele was a 216 bp fragment (Fig. 2B). Tissues were homogenized with water at 20% of concentration by weight in an all-glass Potter–Elvehjem homogenizer. Initial Semiquantitative RT–PCR extraction with chloroform-methanol was done as described previously (6). Brain, kidney and testis lipids were fractionated Whole brain tissues from saposin A–/–, saposin A+/–, wild-type to neutral and acidic fractions using the reverse phase column and total saposin-deficient mice were collected. The mice were essentially according to Kyrklund (38) (Varian Bond Elute, C-18; killed by decapitation and the brains were rapidly dissected 3 ml/500 mg). This procedure gave the advantage of clean and processed for RNA extraction. Total RNA was isolated separation of all gangliosides and sulfatide into the acidic fraction, from each whole brain using TRI reagent (Sigma) following simple and rapid desalting and no need for saponification of the manufacturer’s protocol. Total RNA (2.5 µg) was reverse- the acidic fraction. Aliquots of the brain neutral lipid fraction transcribed using the SuperScript first strand cDNA synthesis were subjected to the mercuric chloride-saponification procedure system (Gibco BRL) and oligo (dT)12–18 primers. The cDNA in order to remove essentially all glycerophospholipids (39). was then amplified using specific primers for mouse saposin and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Quantitation of psychosine as an internal control. The primers used were: sense, 5′-CTGT- CGGCCTCGTGCAAGGAGGTGGTT-3′;antisense,5′-GGCA- Brain psychosine (galactosylsphingosine) was determined by GTCTCCATGTTCTGACAC-3′ for mouse prosaposin [1323 bp the HPLC procedure (40,41), as modified by us more recently (exons 4–14)]; and sense, 5′-CCATGGAGAAGGCCGGGG-3′; (42), except that the mobile phase was methanol-5 mM sodium antisense, 5′-CAAAGTTGTCATGGATGACC-3′ for mouse phosphate buffer, pH 7.0 (89:11) with 50 mg/l sodium octylsulfate GAPDH (35). The optimized numbers of PCR cycles allowing as a ion-pairing agent (16). The peaks of phytosphingosine, the signal to be in the linear portion of the amplification curve psychosine (galactosylsphingosine), sphingosine, sphinganine were 20 cycles for prosaposin and 17 cycles for GAPDH. A and eicosasphinganine (internal standard) were eluted in this negative control lacking template cDNA was included in each order and were cleanly separated from each other and from RT–PCR. For quantitative analysis, the stained gel was other interfering fluorescent materials. Glucosylsphingosine scanned with an image scanner (ScanJet ADF, Hewlett- and galactosylsphingosine were eluted with the same retention Packard) and the band densities were digitized using the auto- time in this HPLC system. However, our experience with mated digitizing software (UN-SCAN-IT gel version 5.1, Silk GalCer synthase-deficient mice indicates that mouse brain Scientific). The values obtained for prosaposin bands were contains no detectable amount of glucosylsphingosine, with normalized for the GAPDH bands (Fig. 2C). 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 Animal care and clinical studies expressed in pmol/mg tissue protein. The data were evaluated All animal protocols used in these studies were approved by by the unpaired Student’s t-test. the Internal Review Board of our university. Mice were maintained with access to water ad libitum. All mice were closely ACKNOWLEDGMENTS observed throughout their lives. Body weight was recorded daily as an objective parameter for development and progression The authors thank Dr Nobuyo Maeda for providing us with the of the disease. In order to determine the natural course of the vector (OSdupdel), Dr Randy J. Thresher in the University of disease, some mice were allowed to live as long as they could North Carolina Animal Models Core Facility for helping with be maintained humanely according to the acceptable practice ES cell handling, Ms Elise Cash and Clarita Langaman for the of laboratory animal care but without forced feeding or other tissue preparation and Mr Joe Langaman for cell culture and extraneous interventions. For biochemical and pathological other technical assistance. This work was supported in part by evaluation, some mice were killed at ∼1, 2, 4 and 5 months. research grants, RO1-NS24289 and a Mental Retardation Human Molecular Genetics, 2001, Vol. 10, No. 11 1199

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