Targeted disruption of the mouse sphingolipid activator protein gene: a complex phenotype, including severe leukodystrophy and wide-spread storage of multiple sphingolipids
Targeted disruption of the mouse sphingolipid activator protein gene: a complex phenotype, including severe leukodystrophy and wide-spread storage of multiple sphingolipidsNobuya Fujita1, Kinuko Suzuki1,2, Marie T. Vanier6, Brian Popko1,3,4, Nobuyo Maeda2,4, Andreas Klein7, Margarete Henseler7, Konrad Sandhoff7, Hiroyuki Nakayasu1 and Kunihiko Suzuki1,5,*
1Brain and Development Research Center, 2Department of Pathology and Laboratory Medicine, 3Department of Biochemistry and Biophysics, 4Program in Molecular Biology and Biotechnology, 5Departments of Neurology and Psychiatry, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA, 6INSERM-CNRS 189, Lyon-Sud School of Medicine and Fondation Gillet-Mérieux, Lyon-Sud Hospital, F-69921 Oullins Cedex, France and 7Institut für Organische Chemie und Biochemie, Universität Bonn, D-53121 Bonn, Germany
Received February 21, 1996;Revised and Accepted March 16, 1996
The four established or putative sphingolipid activator proteins derive from a large precursor protein encoded by a single gene. In addition to generating the four sphingolipid activator proteins, the precursor protein is suspected of having functions of its own, as, for example, a lipid binding/transport protein or a neurotrophic factor. The gene also appears to encode the Sertoli cell major sulfated glycoprotein. Sequence similarities have been noted with many other proteins of diverse functions. One patient and a fetus in a single family with a complete defect of this gene due to a mutation in the initiation codon exhibited complex pathological and biochemical abnormalities. Mutant mice homozygous for an inactivated gene of the sphingolipid activator protein precursor exhibit two distinct clinical phenotypes-neonatally fatal and later-onset. The latter develop rapidly progressive neurological signs around 20 days and die by 35-38 days. At 30 days, severe hypomyelination and periodic acid-Schiff-positive materials throughout the nervous system and in abnormal cells in the liver and spleen are the main pathology. Most prominently lactosylceramide, and additionally ceramide, glucosylceramide, galactosylceramide, sulfatide, and globotriaosylceramide are abnormally increased in the brain, liver, kidney, and their catabolism abnormally slow in cultured fibroblasts. Brain gangliosides are generally increased, particularly the monosialogangliosides. The clinical, pathological and biochemical phenotype closely resembles that of the human disease. This model not only allows further clarification of the physiological functions of the four individual sphingolipid activator proteins but also should be useful to explore putative functions of the precursor protein.
The SAP -/- line of mice was generated from one of the targeted Es cell lines, S29. The Southern blot analysis of genomic DNA fragments, generated by either HindIII or BamHI digestion, gave the size distribution expected from the restriction maps of the normal and targeted SAP gene (Fig. 1 ). On the Northern blot analysis, no SAP precursor mRNA could be detected from tissues of affected homozygous mice, while its quantity was reduced in heterozygous carriers, as expected (Fig. 2 ).
Of the first 126 offspring from carrier-to-carrier mating that survived beyond a few days after birth, 36 (29%) mice had the wild-type genotype, 68 (55%) mice were carriers and 16 (13%) were homozygous affected. Genotype determination was inconclusive for three mice. Five of the total nine mice that died within a day or two of birth were SAP -/-. Therefore, a disproportionate percentage of SAP -/- mice appear to die in utero or during the neonatal period. Those SAP -/- mice that survive beyond the first few days feed and grow normally together with their littermates until about 18-20 days when they develop first neurological symptoms. By then, affected mice are slightly smaller than their littermates. Initial symptoms are tremulousness of the head and mild weakness/ataxia of the hind legs. These manifestations progress rapidly during the next ten days to gross shaking of the head and then the trunk, and severe weakness of all legs. After 30 days, intermittent seizures develop and progress to continual tonic status epilepticus. Affected mice die around 35 days in an emaciated general condition. Attempts at keeping affected mice alive beyond 35-38 days by forced feeding have been unsuccessful. By then, the body size of the affected mice is less than 1/2 of their littermates.
The external aspects of the brains of SAP -/- mice appeared grossly normal. However, on coronal sections, the white matter of SAP -/- mouse was pale and ill-defined from the gray matter, suggesting paucity of myelin. No gross abnormalities were noted in the visceral organs. There was no organomegaly. The size of the kidney was significantly smaller in the affected mice (207 +- 31 mg/2 kidneys, n = 4) than in the control mice (253 +- 34 mg/2 kidneys, n = 5), consistent with the smaller bodies of the affected mice.
The primary neuropathology of SAP -/- mouse was that of combined neuronal storage and leukodystrophy. No obvious abnormalities in brain development or the cellular migration and architecture of the central nervous system were noted on the light microscopic level, although detailed morphometric studies have not yet been undertaken. There was neuronal storage in the retinal ganglion cells and in the neurons throughout the brain and spinal cord. The storage material was periodic acid-Schiff (PAS)-negative on paraffin sections and appeared as finely vacuolated (Fig. 3 A) or as coarse eosinophilic granules. However, they were strongly PAS-positive in frozen sections (Fig. 3 B). Finely vacuolated storage neurons were more conspicuous in the cerebral cortex while eosinophilic storage materials were more prominent in the neurons in the entorhinal cortex. In the hippocampus, storage neurons were most abundant in the CA3 region. There were many spherical or ovoid structures containing eosinophilic granular materials, some of which were Bielschowsky-positive. They are most likely distended axons (axonal spheroids) (Fig. 3 C). Scattered PAS-positive cells with small round nuclei identified as macrophages/microglia were present in the white matter (Fig. 3 D), basal ganglia, thalamic nuclei, the cerebellar molecular layer as well as in some areas of the cerebral cortex. The white matter was generally hypomyelinated, most prominently in the corpus callosum and cerebral white matter and least in the spinal cord (Fig. 3 E, F). Myelin ovoids suggesting degeneration of myelin were also noted in the corpus callosum, the internal capsule and the spinal white matter. Spinal roots, and the trigeminal and sciatic nerves showed similar degenerative changes of myelin (Fig. 3 G), although the extent of degeneration was variable. In the cerebellum, PAS-positive macrophages/microglia were noted in the molecular layer as well as in the white matter. Neuronal storage was present in the deep cerebellar nuclei but not apparent in the Purkinje or granular cells, although the soma of some Purkinje cells was vacuolated. Many Bielschowsky stain-positive axonal spheroids were present in the cerebellar white matter. The neuronal storage was also noted in the trigeminal and spinal dorsal root ganglia. Neuronal storage and myelin degeneration were also present in the spinal cord. No sudanophilic or metachromatic materials were found in any of the sections examined.
In the liver, clusters of histiocytes with abundant eosinophilic cytoplasm were seen in the hepatic sinusoid. These cells were only faintly PAS-positive, while hepatocytes were strongly PAS-positive even in paraffin sections (Fig. 3 H). Similar cells were noted in the spleen and lymph nodes. The kidney, heart and lungs appeared normal on the light microscopic level.
Electron microscopic studies revealed that the storage materials in the cerebral cortical neurons consisted of scattered or variously clustered small concentric lamellar and/or electron-dense granular structures. They were often bound by a membrane (Fig. 4 A). Distended axons (axonal spheroids) contained similar concentric or lamellar structures, which, however, were not clustered (Fig. 4 B). Ultrastructural features of the intracellular storage materials in the hepatic sinusoids were more complex than those in neurons. They were composed of concentric outer membranes and short straight or curved membranous or small vesicular inner structures (Fig. 4 C). Some hepatocytes contained electron-dense membrane-bound inclusions, which displayed electronlucent short needle-like inner structures (Fig. 4 D).
Brain lipids (Fig. 5 A; Table 1 ). The most conspicuous specific abnormality in both gray and white matter was a major accumulation of lactosylceramide (more than 50-fold normal), which appeared to account for the strong PAS staining on frozen sections. The chromatographic behavior of lactosylceramide suggested an identical fatty acid composition in gray and white matter. A small but clearly pathological amount (estimated at about 30-50 nmol/g) of another glycolipid migrating as gangliotriaosylceramide was also detected. Due to the small size of the brains analyzed, no attempt was made to quantitate glucosylceramide. By visual inspection of appropriate thin-layer chromatograms, no clear increase of free ceramide could be found in the brain. From visual inspection of the total lipid fraction as well as the quantitative data, the white matter lipids reflected the lack of myelin observed histologically, with reductions of both galactosylceramide and sulfatide to approximately 25% of the normal amounts, together with diminished amounts of cholesterol and phospholipids, particularly ethanolamine phospholipids and sphingomyelin with longer-chain fatty acids. No obvious abnormalities in the levels of cholesterol, glycerophospholipids and sphingomyelin were observed in the gray matter.
The total ganglioside sialic acid was increased significantly in both gray and white matter, more conspicuously in the latter with large relative increases in the monosialogangliosides, GM1, GM2 and GM3 (Fig. 5 B). Their relative increase alone can be interpreted as non-specific. However, because of the substantial increases in the total ganglioside sialic acid, some of the higher gangliosides, most notably GD1a, were also significantly increased on the wet weight or total lipid basis. This finding is unusual and to our knowledge has been described only in an unusual genetic disease occurring in the emu (37 ).
Metabolic studies with cultured fibroblasts (Fig. 7 ). These experiments were done with skin fibroblasts obtained from 30 day old mice. The results essentially corroborated the analytical findings on various organs described above. Increased retention of the radioactive label was found in ceramide, glucosylceramide and lactosylceramide. In addition, GM3-ganglioside retained more radioactivity than the control fibroblasts. Most importantly, the general pattern of sphingolipid abnormalities in the cultured mouse fibroblasts was qualitatively identical with that of the cultured fibroblasts from the human patients with genetic total SAP deficiency.
The Northern blot analysis demonstrated complete absence of SAP mRNA in affected homozygous mice. This finding was particularly important because of the nature of the SAP gene. The targeted SAP gene was interrupted at exon 3. Ordinarily, this would have assured complete loss of the functional translation product. However, the normal SAP mRNA is translated and then processed to generate four small sphingolipid activator proteins. Exon 3 is within the domain of SAP-A and there are several methionine codons downstream of the disrupted site. If the transcript could be processed to a stable mRNA and if any of these downstream ATG could serve as the initiation codon, there is a possibility that a precursor protein truncated at the N-terminus might be synthesized and subsequently processed to generate some of the downstream SAPs with normal sequences. The complete absence of mRNA excludes such a possibility.
Cloning of the mouse SAP gene from the 129 strain. The 2.7 kb mouse cDNA in pGEM-4Z encoding the sphingolipid activator proteins (76 ) was excised from the vector and further digested with EcoRI and SphI to 1.0 and 1.7 kb fragments and subcloned into pUC18 (pSAP-S and pSAP-L). The pSAP-S which spanned exons 2 through 10 was used to screen a genomic library from the mouse 129SV strain in [lambda]-FIX II essentially according to the manufacturer's instructions (Stratagene, La Jolla, CA). Altogether 11 clones were isolated. One of the clones, designated as [lambda]20-1, spanned 15 kb and included exons 2 through 15.Targeted disruption of the endogenous gene. The overall strategy for the targeting was to disrupt exon 3 with the neomycin-resistant gene (Fig. 1 ). Culture of the BK4 cells, a subclone of E14G2a derived from strain 129/Ola mice, electroporation of the NotI-linearized targeting vector and screening for targeted ES cells by double selection with G418 and Ganciclovir were done essentially as previously described (77 -79 ). Southern blot analysis was used for screening for the targeted disruption of the SAP gene in the ES cells. Genomic DNA was digested with either HindIII or BamHI and a 1.7 kb fragment of the gene that included exon 2 was used as the probe (Fig. 1 ). Two positive clones were identified out of 74 screened. They were micoplasm-free and had the karyotype of 60% (S-25) and 100% (S-29), respectively. Cells from the S-29 line were injected into blastocysts. Germline transmitters among the resultant chimeric mice were mated to generate heterozygous mice and subsequently homozygous mice for the disrupted SAP gene. Northern analysis. Northern blot analysis was done by the standard procedures with total cellular RNA isolated with the Trizol LS reagent (Gibco-BRL) either from the whole body of neonatal pups or from the livers of older mice. A subclone of [lambda]20-1 spanning exons 2-10 was used as the probe. A 28S rRNA-specific oligonucleotide was end-labelled with [[gamma]-32P]ATP and used as the probe for the internal control for the quantity of RNA applied to the gel.
Brain, spinal cord, peripheral nerves and visceral organs from a 30 day old SAP -/- and littermate control mice, and selected tissues (spinal cord, eye, trigeminal and spinal dorsal root ganglions, trigeminal and sciatic nerves) from two 30 day old SAP -/- mice, were examined histologically.
One each of SAP -/- and SAP +/+ mice was anesthetized and perfused with 4% paraformaldehyde. Brains, spinal cord, nerves and various visceral organs were processed for paraffin and plastic embedding. Portions of the cerebrum, cerebellum, spinal cord, eye, dorsal root ganglia and trigeminal and sciatic nerves were cryoprotected by immersion in 0.1 M sodium phosphate buffer (pH 7.4) containing 30% sucrose, quick-frozen in cooled isopentane and sectioned at 7 [mu]m thick with a cryostat. Tissues from two 30 day old SAP -/- mice that were killed for biochemical analysis were immersion-fixed in situ for a few days, tissues removed and processed for paraffin embedding.
The paraffin sections of the CNS and PNS tissues were stained with hematoxylin and eosin, solochrome and eosin, luxol fast blue/periodic acid and Schiff (LFB-PAS) and with the Bielschowsky stains. The systemic organs were stained with hematoxylin and eosin, and with periodic acid/Schiff (PAS). In order to evaluate the degree of myelination, paraffin sections of the cerebrum, cerebellum and spinal cord were processed for immunocytochemical detection of the myelin basic protein with the SMI 99 antibody diluted 1:1 000 (Sternberger Monoclonals Inc., Bethesda, MD) using the Autoprobe III kit with streptavidin-conjugated peroxidase base (Biomed Co., Foster City, CA). Frozen sections of the CNS and PNS tissues were stained with PAS, Sudan IV, toluidine blue and acidic cresyl violet.
Plastic-embedded tissues were sectioned in 1 [mu]m thickness and stained with toluidine blue. Selected areas of the cerebrum and liver were further processed for thin sectioning, stained with uranyl acetate and lead citrate and examined with a Zeiss 10A electron microscope.
Samples. The brain, liver and kidney obtained from four affected and five control mice, all 30 day old, were included in this series of the first characterization. Initial lipid and enzyme analyses were all performed separately on specimens from individual mice. The five control mice included three wild-type and two heterozygous carriers. In no instances were there any differences between the wild-type and heterozygous mice and the results of the control group were treated together for statistical evaluation. For specific analytical purposes, lipid extracts from some samples were pooled at later stages as indicated. Brains were dissected into gray and white matter. Bilateral kidneys were used together for each mouse without further dissection, while appropriate portions of the liver were dissected for the liver samples. The ranges of the samples used for analyses were 54-157 mg (gray matter), 38-92 mg (white matter), 361-490 mg (liver) and 166-286 mg (kidneys).General lipid extraction. The initial extraction was based on the method of Svennerholm and Fredman (80 ) as described in detail by Svennerholm et al. (81 ). The tissue was homogenized with 0.6 ml of water in an all-glass Potter-Elvehjem homogenizer and extracted by addition of 2 ml of methanol and 1 ml of chloroform. After at least 15 min, the extract was centrifuged at 1 000 g for 10 min and the supernate removed to a small round bottom flask. The pellet was resuspended in 0.7 ml water and re-extracted with 3 ml of chloroform-methanol, 1:2 (v/v). After centrifugation, the combined supernates were taken to dryness in a rotary evaporator and the lipids dissolved in 5 ml of chloroform-methanol-water (60:30:4.5, v/v/v). The extract was desalted through a 1 g Sephadex G-25 Fine column (82 ).Separation of gangliosides from other lipids. Gangliosides were separated from other lipids on a 1 g silica gel column (Silica gel 60, 230-400 mesh, Merck A.G., Darmstadt, FRG) packed in chloroform (Svennerholm et al. 1991). The lipid sample was redissolved in 2 ml of chloroform-methanol (9:1, v/v), applied to the column, the flask rinsed and applied to the column with two portions of 1 ml of the same solvent. The flask was finally rinsed with 2 ml of chloroform-methanol-water (65:25:4, v/v/v) which was also added to the column. Finally, the column was eluted with additional 6 ml of chloroform-methanol-water (65:25:4, v/v/v). All the eluates to this point were pooled and designated as the `non-ganglioside lipid fraction'. Then the `ganglioside fraction' was eluted with additional 10 ml of chloroform-methanol-water (3:6:2, v/v/v). Test runs and thin-layer chromatography indicated that this procedure cleanly separated all non-ganglioside lipids including sphingomyelin into the first fraction and all gangliosides including GM3 into the ganglioside fraction without cross-contamination.Removal of glycerophospholipids from the non-ganglioside lipid fraction. Aliquots of the brain `non-ganglioside lipid fraction' were subjected to the mercuric chloride-saponification procedure essentially according to Abramson et al. (83 ). After the alkaline hydrolysis step, the solutes were partitioned, the upper phase discarded and the lower phase washed twice more. The final lower phase was evaporated to dryness and dissolved in a known volume of chloroform-methanol-water (60:30:4.5, v/v/v). All glycerolipids including plasmalogens abundant in the brain were completely degraded and removed by this procedure, leaving cholesterol, cholesteryl esters, liberated free fatty acids, and sphingolipids. The mercuric chloride step was omitted for the liver and kidney lipids since they contain little plasmalogens. Separation of kidney sulfatide and GM3-ganglioside. Pooled aliquots from kidney lipids (corresponding to 100 mg tissue from each mouse) were used to evaluate specifically the state of monohexosylceramides, sulfatides and GM3-ganglioside. Sulfatides and gangliosides were isolated by anion-exchange chromatography on a 0.1 g column of DEAE-Sephadex A-25 in acetate form (84 ) packed in C-M-W (30:60:5, v/v/v). The sample was dissolved in 2 ml of the same solvent, applied to the column and washed with 6 ml of the same solvent. This pass-through fraction contained all of the neutral sphingolipids. Acidic lipids were eluted with 8 ml of C-M-0.8 M potassium acetate (30:60:5, v/v/v). This fraction containing sulfatide and gangliosides was saponified and desalted on a Sephadex G-25 column as described above.Quantitation of lipids. The total lipid phosphorus, sphingomyelin and cholesterol were determined on the `non-ganglioside lipid fraction' as previously described (85 ). The total sialic acid content of the ganglioside fraction was determined by the resorcinol method (86 ,87 ) with N-acetylneuraminic acid as the standard. Thin-layer chromatography was done with the high-performance thin-layer plates (HPTLC 60, Merck) developed in appropriate solvents: double-development in chloroform-methanol-water (65:25:4, v/v/v) followed by hexane-ethylacetate-acetic acid (90:10:1,v/v/v) for general lipid visualization, chloroform-methanol-acetic acid (47:1:2, v/v/v) for ceramide, and chloroform-methanol-0.2% CaCl2 (55:45:10, v/v/v) for gangliosides. Glucosylceramide and galactosylceramide were separated on borate-impregnated thin-layer plates (88 ). Lipids were visualized by copper acetate-phosphoric acid (general), orcinol-sulfuric acid (carbohydrate) or resorcinol (gangliosides) (86 ) sprays and heating. For quantitation, samples were applied with a CAMAG Linomat IV apparatus together with appropriate calibrated lipid standards and densitometry of the plates performed using a CAMAG TLC scanner model II operated with the PC-CATS 3 evaluation software. Special care was taken to keep densities of the bands within the linear range with respect to the quantity of the lipid. For this series of initial characterization, lipids were identified primarily by their TLC mobility compared with authentic lipid standards prepared in the laboratory from normal and pathological human tissues. Because of the small sizes of the starting specimens and of the multiple analytical steps, the recovery of glycolipids present in small quantities are not expected to be quantitative. However, such losses during the analytical procedures should be no more than 30% for the brain and liver samples and should be consistent among all samples. Even in the kidney, where the loss can be even greater, the expected consistent losses among samples allow meaningful comparison between the affected mice and the controls. Recoveries of the major lipids, such as cholesterol, total phospholipids, sphingomyelin and gangliosides should be essentially quantitative.
Assays for activities of the lysosomal enzymes were done with the liver from the same mouse specimens as used for lipid analyses. Total homogenates (2-4 mg protein/ml) prepared in ice-cold distilled water and subjected to 3 cycles of freeze-thawing/1 min sonication in a water bath-type sonicator (Branson Ultrasonic Corp., Danbury, CT) were used as the enzyme source. Activities of total [beta]-hexosaminidase, [beta]-galactosidase, [beta]-glucosidase and [alpha]-galactosidase were assayed with fluorogenic 4-methylumbelliferyl substrates according to the procedures routinely used for diagnosis of patients with sphingolipidoses (89 ). The assay system for [beta]-glucosidase with 4-methylumbelliferyl [beta]-glucoside as the substrate included sodium taurocholate to measure specifically that portion of [beta]-glucosidase that represents glucosylceramidase (90 ,91 ). Micromethods with radioactively labelled natural lipid substrates were used for assays of glucosylceramidase, acid sphingomyelinase and galactosylceramidase (89 ,92 ,93 ). Arylsulfatase A activity was determined with p-nitrocatechol sulfate on the pH 5.0 supernatant at 0oC to minimize the interference from arylsulfatases B and C (94 ). The content of protein was determined by a modified Lowry procedure with bovine serum albumin as the standard (95 ).
Fibroblast cultures were established from 30 day old wild-type and affected mice with the standard procedure of our laboratory. The life span of mouse fibroblasts was relatively limited when the culture was started at such a late age. Cells tended to divide rather slowly and then remained quiescent after several divisions. Normal human fibroblasts were from the Johanniter Children's Hospital, Sankt Augustin, Germany. The SAP-precursor-deficient fibroblasts from the patient who died at 16 weeks were kindly provided by Prof. Harzer (Universität Tübingen, Germany), and have been described before (24 ,28 ,29 ). Cells were cultured as described by Weitz et al. (96 ). The metabolic studies were performed with confluent cells in 21 cm2 plastic culture dishes. Sphingolipids of cultivated fibroblasts were labeled with L-[3-14C]serine for 24 h, chased for 120 h, extracted and their radioactivity determined after thin-layer chromatographic separation essentially as described previously (51 ).
The mouse sphingolipid activator cDNA (76 ) used as the initial probe to isolate the gene from the genomic library of the 129 strain was kindly provided by Prof. Kitagawa and Dr Sakiyama, Tokyo, Japan. This work has been supported in part by research grants, RO1-NS24289, RO1-24453 and RO1-NS28997, and a Mental Retardation Research Center Core Grant, P30-HD03110, from the USPHS, a research grant from the ELA to M. T. Vanier and a grant, SFB 284, from the Deutsche Forschungsgemeinschaft to K. Sandhoff.
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*To whom correspondence should be addressed at: Brain & Development Research Center, CB#7250, 311 BDRC, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7250, USA
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