Rumpshaker-like proteolipid protein (PLP) ratio in a mouse model with unperturbed structural and functional integrity of the myelin sheath and axons in the central nervous system.

The gene plp on the X chromosome encodes the isoforms proteolipid protein (PLP) and DM(20), two dominant integral membrane proteins of central nervous system (CNS) myelin. DM(20) results from the activation of the cryptic splice site in exon III of the PLP gene. We inserted a sense-orientated loxP flanked neomycin-gene into intron III of the plp sequence, using homologous recombination in embryonic stem cells and generated the homozygous neoS mouse line. Unlike the previously described complete PLP/DM(20) ablation (plp(-/-)), which has been obtained by introducing a neo-gene in antisense-orientation in the same position of intron III, the plp expression surprisingly revealed reduced mRNA levels. The PLP isoform was reduced to 50%, but DM(20) expression was unaffected. This protein pattern resembles the expression profile of the PLP isoforms in the natural occurring rumpshaker mutant. Electron microscopic examination revealed a normal compaction of CNS-myelin and maintenance of axon integrity. PLP expression levels of the wt control were recovered by Cre excision of the neo-selection gene after intercrossing neoS mice and oligodendrocyte-specific Cre-mice. These data strongly hint at different functions of intron III in PLP/DM(20)-specific splicing and mRNA stability. Furthermore evidence is provided for functionally affected translation products of the PLP gene in the rumpshaker mutant, whereas no PLP-isoform occur in plp(-/-) mice generated by introducing a selectable marker into intron III in antisense orientation.

Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains.

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.

Early onset of axonal degeneration in double (plp-/-mag-/-) and hypomyelinosis in triple (plp-/-mbp-/-mag-/-) mutant mice.

Double (plp-/-mag-/-) and triple (plp-/-mbp-/-mag-/-) null-allelic mouse lines deficient in proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) were generated and characterized genetically, biochemically, and morphologically including their behavioral capacities. The plp-/-mag-/- mutant develops a rapidly progressing axon degeneration in CNS with severe cognitive and motor coordinative deficits but has a normal longevity. CNS axons of the plp-/-mbp-/-mag-/- mouse are hypomyelinated and ensheathed by "pseudomyelin" with disturbed protein and complex lipid composition. The shiverer trait in the plp-/-mbp-/-mag-/- similar to the plp-/-mbp-/- mutant is significantly ameliorated, and its lifespan is considerably prolonged. The longevity of these dysmyelinosis mouse mutants recommends them as suitable models for the long-term evaluation of stem cell therapeutic strategies.

Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase.

The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular data on the neutral sphingomyelinases (nSMases). We recently identified a mammalian nSMase, an integral membrane protein with remote similarity to bacterial sphingomyelinases. However, its ubiquitous expression pattern is in contrast to previous findings that sphingomyelinase activity is found mainly in brain tissues. By using an improved database search method, combined with phylogenetic analysis, we identified a second mammalian nSMase (nSMase2) with predominant expression in the brain. The sphingomyelinase activity of nSMase2 has a neutral pH optimum, depends on Mg(2+) ions, and is activated by unsaturated fatty acids and phosphatidylserine. Immunofluorescence reveals a neuron-specific punctate perinuclear staining, which colocalizes with a Golgi marker in a number of cell lines. The likely identity of nSMase2 with cca1, a rat protein involved in contact inhibition of 3Y1 fibroblasts, suggests a role for this enzyme in cell cycle arrest. Both mammalian nSMases are members of a superfamily of Mg(2+)-dependent phosphohydrolases, which also contains nucleases, inositol phosphatases, and bacterial toxins.

Perturbation of membrane microdomains reduces mitogenic signaling and increases susceptibility to apoptosis after T cell receptor stimulation.

Acid sphingomyelinase-deficient (asmase-/-) mice generated by gene targeting abundantly store sphingomyelin in the reticuloendothelial system of liver, spleen, bone marrow, and in brain. Liver cells of asmase-/- mice accumulate sphingomyelin and glycosphingolipids in purified lipid bilayers of microsomes, Golgi, and the plasma membrane, but cholesterol is depleted in the plasma membrane. Detergent-insoluble glycolipid-enriched membrane microdomains (GEM) can be isolated from hepatocytes, embryonic fibroblasts, and splenocytes of wild-type, but not of asmase-/- mice, by sucrose gradient density centrifugation. Lck and other Src-family kinases are reduced in isopycnic fractions of asmase-/- splenocytes compared to GEM-containing fractions of wild-type cells. The proliferation of asmase-/- T lymphocytes is reduced, whereas their susceptibility to Fas-induced apoptosis is increased after T cell receptor (TCR) stimulation. TNF receptor I signaling remains unimpaired. The perturbation of GEM impairs tyrosine phosphorylation and, consequently, mitogenic signaling of the TCR. Reduced MAPK activity-dependent FLICE-like inhibitory protein (FLIP) expression in asmase-/- T lymphocytes increases their sensitivity towards Fas-mediated apoptosis.

Characterization and subcellular localization of murine and human magnesium-dependent neutral sphingomyelinase.

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, an essential lipid constituent of the plasma membrane, lysosomal membranes, endoplasmic reticulum, and the Golgi membrane stacks of mammalian cells. In this study, we report the biochemical and functional characterization and subcellular localization of magnesium-dependent nSMase1 from overexpressing human embryonic kidney (HEK293) cells. Site-directed mutagenesis of conserved residues probably involved in the enzymatic sphingomyelin cleavage as well as the removal of one or both putative transmembrane domains lead to the complete loss of enzymatic activity of human nSMase1 expressed in HEK293 cells. Polyclonal antibodies raised against recombinant mammalian nSMase1 immunoprecipitated and inactivated the enzyme in membrane extracts of overexpressing HEK293 cells and different murine tissues. Cell fractionation combined with immunoprecipitation studies localized the nSMase1 protein predominantly in the microsomal fraction. The enzyme colocalized with marker proteins of the endoplasmic reticulum and the Golgi apparatus in immunocytochemistry. Anti-nSMase1 antibodies did not affect the nSMase activity in the plasma membrane fraction and membrane extracts from murine brain. Our study leads to the conclusion that nSMase1 is one of at least two mammalian neutral sphingomyelinases with different subcellular localization, tissue specificity, and enzymatic properties.

Functional analysis of acid and neutral sphingomyelinases in vitro and in vivo.

The molecular cloning and the elucidation of the gene structures of the acid (aSMase) and a neutral sphingomyelinases (nSMase) of mouse and human facilitated the structural and functional analysis of these enzymes responsible for the catabolism of sphingomyelin present ubiquitously in the membrane lipid bilayer of mammalian cells. The protein and enzymic properties of the glycoprotein aSMase and of a non-glycosylated nSMase residing in the membranes of the endoplasmic reticulum have been analysed in the native as well as in the recombinant shingomyelinases. Important insight was gained from gene targeting experiments in which an aSMase deficient mouse line was generated which mimics the neurovisceral form of the human Niemann-Pick disease. The availability of the cloned aSMase and nSMases discovered so far led to a genetic approach to the verification of the concept that these enzymes in the 'sphingomelin cycle' are responsible for the generation of ceramide regarded as a lipophilic second messenger in the intracellular signal cascades activated by e.g. TNF-alpha, Fas ligand or cellular stress. All the available evidence derived from the aSMase deficient mouse line and several cell lines overexpressing aSMase and nSMase questions a role of ceramide released by the mammalian sphingomyelinases known so far in intracellular signal transduction.

Cotranslational integration of myelin proteolipid protein (PLP) into the membrane of endoplasmic reticulum: analysis of topology by glycosylation scanning and protease domain protection assay.

The four transmembrane domain topology of the proteolipid protein (PLP) in the myelin membrane of the central nervous system (CNS) has been further substantiated by biochemical studies. We have analyzed the cotranslational polytopic integration of nascent PLP during protein synthesis into the membrane of the endoplasmic reticulum (ER) on two routes. Consensus sequences for N-glycosylation were introduced by site directed mutagenesis into the PLP sequence as reporter sites, which upon glycosylation monitor the intraluminal location of the respective domains corresponding to the extracellular side of the plasma membrane. Single, double, and triple mutant cDNAs were constructed for transcription/translation in vitro in the presence of ER-membranes. The glycosylation pattern of the translation products revealed that hydrophilic extramembrane regions 2 and 4 (EMR2/EMR4) and EMR3 of PLP are exposed on opposite sides of the ER membrane. Their localization either at the cytosolic or luminal side of the ER membrane leads to two different topologies. The two modes of membrane integration during in vitro cotranslational translocation were confirmed by protease protection assays with wild-type and truncated PLP polypeptides with either one, two, or three putative transmembrane domains integrated into the ER-membrane. The fragment pattern of the [35S]methionine- or [3H]leucine-labeled polypeptides revealed that EMR3 and EMR4 were exposed with opposite orientation either on the cytosolic or luminal side of the ER membrane supporting the 4-transmembrane helix (TMH) N(in) model with the N and C termini on the cytoplasmic side, as established for the myelin membrane (plasma membrane); the other inversely integrated PLP constructs indicate the 4-TMH-Nout profile. These results are discussed with regard to the PLP biogenesis and the plasma membrane topology in PLP-expressing cells.

Galactosphingolipids and axono-glial interaction in myelin of the central nervous system.

The myelin of central and peripheral nervous system of UDP-galactose-ceramide galactosyltransferase deficient mice (cgt-/-) is completely depleted of its major lipid constituents, galactocerebrosides and sulfatides. The deficiency of these glycolipids affects the biophysical properties of the myelin sheath and causes the loss of the rapid saltatory conduction velocity of myelinated axons. With the onset of myelination, null mutant cgt-/- mice develop fatal neurological defects. CNS and PNS analysis of cgt-/- mice revealed (1) hypomyelination of axons of the spinal cord and optic nerves, but no apoptosis of oligodendrocytes, (2) redundant myelin in younger mice leading to vacuolated nerve fibers in cgt-/- mice, (3) the occurrence of multiple myelinated CNS axons, and (4) severely distorted lateral loops in CNS paranodes. The loss of saltatory conduction is not associated with a randomization of voltage-gated sodium channels in the axolemma of PNS fibers. We conclude that cerebrosides (GalC) and sulfatides (sGalC) play a major role in CNS axono-glial interaction. A close axono-glial contact is not a prerequisite for the spiraling and compaction process of myelin. Axonal sodium channels remain clustered at the nodes of Ranvier independent of the change in the physical properties of myelin membrane devoid of galactosphingolipids. Increased intracellular concentrations of free ceramides do not trigger apoptosis of oligodendrocytes.

Cloned mammalian neutral sphingomyelinase: functions in sphingolipid signaling?

Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, is released by either acid sphingomyelinase or neutral sphingomyelinase (nSMase) and has emerged as a potential lipid signaling molecule. nSMase is regarded as a key enzyme in the regulated activation of the "sphingomyelin cycle" and cell signaling. We report here the cloning, identification, and functional characterization of murine and human nSMase, a ubiquitously expressed integral membrane protein, which displays all established properties of the Mg2+-dependent nSMase of the plasma membrane. Stably nSMase-overexpressing U937 and human embryonic kidney cell lines have been generated for the study of the role of nSMase in signal transduction pathways. Their stimulation by tumor necrosis factor alpha leads only to a moderately elevated ceramide concentration. Activation of Jun kinase and NFkappaB and poly(ADP-ribose) polymerase cleavage are identical in mock- and nSMase-transfected cells. Tumor necrosis factor alpha triggers the ERK1 pathway in none of the cell lines. The cloned nSMase will facilitate further controlled experiments aiming at the definition of a possible role of ceramide as signal transduction molecule.

Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice.

Ceramide generated by lysosomal acid sphingomyelinase (aSMase) has been proposed to contribute to CD28 co-stimulatory signaling pathways. We used an aSMase-deficient mouse line (asmase-/-) to elucidate the role of the aSMase in splenocytes stimulated with either a combination of anti-CD3 and anti-CD28 antibodies, the lectin concanavalin A (Con A) or the superantigen staphylococcal enterotoxin B. All stimuli were shown to induce IL-2 expression, Con A additionally triggered the expression of high-affinity IL-2 receptor. However, in asmase-/- mice secretion of IL-2 was significantly reduced, whereas the intracellular IL-2 levels were elevated. Proliferation of anti-CD3/anti-CD28 or Con A-stimulated aSMase-deficient splenocytes was reduced up to 50% after 72 h in comparison to wild-type cells. We conclude that ceramide generated by aSMase is not involved in CD28 signal transduction, but rather a perturbation of the secretory system is responsible for the impaired proliferation of aSMase-deficient splenocytes.

Composition and biophysical properties of myelin lipid define the neurological defects in galactocerebroside- and sulfatide-deficient mice.

Oligodendrocytes and Schwann cell-specific proteins are assembled with a highly ordered membrane lipid bilayer to the myelin sheath of axons, which functions as an insulator and allows rapid saltatory conduction. We approached the question of the function of the CNS and PNS myelin-specific galactospingolipids cerebrosides and sulfatides by generating a ceramide galactosyltransferase null allelic mouse line (cgt-/-). Galactocerebroside- and sulfatide-deficient myelin loses its insulating properties and causes a severe dysmyelinosis that is incompatible with life. Here, we describe the biochemical and biophysical analysis of the myelin lipid bilayer of cgt-/- mice. The lipid composition of CNS and PNS myelin of cgt-/- mice is seriously perturbed and the sphingolipid biosynthetic pathway altered. Nonhydroxy and hydroxy fatty acid-substituted glycosylceramides (GlcC) are synthesized by oligodendrocytes and sulfated GlcC in addition in Schwann cells. The monogalactosyldiglyceride fraction is missing in the cgt-/- mouse. This new lipid composition can be correlated with the biophysical properties of the myelin sheath. The deficiency of galactocerebrosides and sulfatides leads to an increased fluidity, permeability, and impaired packing of the myelin lipid bilayer of the internodal membrane system. The loss of the two glycosphingolipid classes causes the breakdown of saltatory conductance of myelinated axons in the cgt-/- mouse.

Human and murine serine-palmitoyl-CoA transferase--cloning, expression and characterization of the key enzyme in sphingolipid synthesis.

Serine palmitoyltransferase (SPT, EC 2.3.1.50) is the key enzyme in sphingolipid biosynthesis. It catalyzes the pyridoxal-5'-phosphate-dependent condensation of L-serine and palmitoyl-CoA to 3-oxosphinganine. Human expressed-sequence-tag (EST) clones are similar to the two yeast genes for synthesis of long-chain bases, LCB1 and LCB2, which are believed to encode two subunits of SPT [Buede, R., Pinto, W. J., Lester, R. L. & Dickson, R. C. (1991) J. Bacteriol. 173, 4325-5332; Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L. & Dickson, R. C. (1994) Proc. Natl Acad. Sci. USA 91, 7899-7902]. We have cloned and characterized two complete human and murine cDNA sequences named hLCB1 & mLCB1 and hLCB2 & mLCB2, respectively, similar to the yeast LCB1 and LCB2 genes. Human embryonic kidney cells (HEK 293) transfected with murine sequences of LCB1 (mLCB1) and LCB2 (mLCB2) independently and in coexpression showed an overexpression of the transcripts on the mRNA and protein level. The enzymatic activity of cells expressing mLCB2 alone or coexpressed with mLCB1 was three times higher than the activity of untransfected HEK cells. mLCB1 expression was not required for the synthesis of 3-oxo-sphinganine in mammalian cells. Transcription/translation in vitro yielded mLCB1 (53 kDa) and mLCB2 (63 kDa). The two proteins do not contain a signal peptide nor are they glycosylated. The endogenous and overexpressed SPT activity were both sensitive to common SPT inhibitors. Labeling studies with [1-(14)C]palmitic acid indicated that cell lines transfected with mLCB2 preferentially use the excess sphingoid bases for glucocerebroside and galactocerebroside synthesis. Our results provide conclusive genetic and biochemical evidence that the human and murine LCB2 genes described here encode serine palmitoyltransferase. Further studies will be required to unravel the function of the LCB1 gene in mammalian cells.

Functional analysis in vivo of the double mutant mouse deficient in both proteolipid protein (PLP) and myelin basic protein (MBP) in the central nervous system.

Myelination is an important developmental process of the central (CNS) and peripheral nervous system (PNS). To unravel the functions of the two dominant myelin proteins in the CNS, proteolipid protein (PLP) and myelin basic protein (MBP), we generated and characterized the homozygous double mutant mouse line (plp-/-, mbp-/-), which is viable and fertile. Plasma membrane processes of oligodendrocytes deficient in PLP and MBP, but not in myelin-associated glycoprotein (MAG), spirally wrap large diameter axons, tightly adhering at their extracytosolic surfaces and forming a pseudo-compacted myelin. Neuromotor activity and coordination are considerably improved compared to the shiverer trait.

Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration.

Four L-glutamate neurotransmitter transporters, the three Na(+)-dependent GLAST-1, GLT-1 and EAAC-1, and the Cl(-)-dependent EAAT-4, form a new family of structurally related integral plasma membrane proteins with different distribution in the central nervous system. They may have pivotal functions in the regulation of synaptic L-glutamate concentration during neurotransmission and are believed to prevent glutamate neurotoxicity. To investigate the specific physiological and pathophysiological role of the neuronal EAAC-1, which is also expressed in kidney and small intestine, we have generated two independent mouse lines lacking EAAC-1. eaac-1(-/-) mice develop dicarboxylic aminoaciduria. No neurodegeneration has been observed during a period of >12 months, but homozygous mutants display a significantly reduced spontaneous locomotor activity.

Tumor necrosis factor alpha activates NF-kappaB in acid sphingomyelinase-deficient mouse embryonic fibroblasts.

Tumor necrosis factor alpha (TNF-alpha) is one of the most potent inducer of the nuclear transcription factor kappaB (NF-kappaB). Activation of NF-kappaB is initiated by phosphorylation of the inhibitory subunit of the IkappaB-alpha-NF-kappaB complex. This leads to the dissociation of the complex and degradation of IkappaB-alpha. NF-kappaB is translocated into the nucleus. The sphingomyelin pathway is thought to mediate the TNF-alpha-induced activation of NF-kappaB by its second messenger ceramide. We have used the recently established acid sphingomyelinase-deficient mouse line (asmase-/- mice) to evaluate the role of acid sphingomyelinase in the TNF-alpha-induced signal transduction pathway. Here we present experimental evidence that acid sphingomyelinase is not involved in the TNF-alpha-induced activation of NF-kappaB. TNF-alpha treatment induced the dissociation and degradation of IkappaB-alpha and the nuclear translocation of NF-kappaB in embryonic fibroblasts derived from asmase-/- and wild type mice indiscriminately.

Human mitochondrial enoyl-CoA hydratase gene (ECHS1): structural organization and assignment to chromosome 10q26.2-q26.3.

The second step in mitochondrial fatty acid beta-oxidation is catalyzed by short chain enoyl-CoA hydratase (ECHS1; EC 4.2.1.17). Inherited disorders of this pathway of energy metabolism present clinical and laboratory features resembling sudden infant death syndrome and Reye-like syndrome. To investigate the role of ECHS1 further, the gene structure was analyzed and its chromosomal locus determined. A fragment of rat liver ECHS1 cDNA was employed for isolation and characterization of two overlapping genomic clones encompassing the entire human ECHS1 gene. The gene, approximately 11 kb, is composed of eight exons, with exons I and VIII containing the 5'- and 3'-untranslated regions, respectively. Two major transcription start sites, located 62 and 63 bp upstream of the translation initiation codon, were mapped by primer extension analysis. The immediate 5'-flanking region of the ECHS1 gene is GC-rich and contains several copies of the SP1 binding motive but no typical TATA or CAAT boxes are apparent. Alu repeat elements have been identified within the region -1052/-770 relative to the cap site and in intron 7. The human ECHS1 gene locus was assigned to chromosome 10q26.2-q26.3 by fluorescence in situ hybridization.

Inhibition of the high-affinity brain glutamate transporter GLAST-1 via direct phosphorylation.

Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This downregulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [32P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [35S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.

Light and electron microscopic analysis of the central and peripheral nervous systems of acid sphingomyelinase-deficient mice resulting from gene targeting.

The acid sphingomyelinase (aSmase)-deficient mouse line recently generated by gene targeting (Otterbach and Stoffel, 1995) develops a lethal storage disease which is phenotypically comparable to the neurovisceral form of the human sphingomyelinosis, Niemann-Pick disease type A (NPA). This report describes the progressive accumulation of uncatabolized lipid substrates at the cellular and ultrastructural level in different regions of the nervous system of homozygous aSmase-/- mice, including cerebrum, cerebellum, spinal cord, optic nerve and peripheral nerves. We saw a cytoplasmic accumulation of pleomorphic lysosomal structures in cells of all regions under study, most extensively in macrophages, vascular endothelial cells, and also in neuronal perikarya. The complete and early degeneration of Purkinje cells was particularly striking. Moreover, we found a storage material in the cytoplasm of Schwann cells and to a minor extent in oligodendrocytes. In most advanced stages of the disorder, we detected an axonal dystrophy in both the central nervous system (CNS) and peripheral nervous system (PNS), without signs of dysmyelination or demyelination. The morphological changes of the central and peripheral nervous systems in the homozygous aSmase-/- mouse line closely resemble those in human NPA.

Pathology of visceral organs and bone marrow in an acid sphingomyelinase deficient knock-out mouse line, mimicking human Niemann-Pick disease type A. A light and electron microscopic study.

A recently generated aSmase knock-out mouse line develops a lethal storage disease which mimics the neurovisceral form of Niemann-Pick disease in man. In extension to the previously described neuropathological changes, the purpose of this study was to provide a detailed morphological, particularly ultrastructural analysis of the visceral organs of these animals including spleen, liver, intestine, lung, and kidney along with a sequential histological investigation of the bone marrow. Our results showed a progressive lysosomal storage as indicated by an increasing amount of foam cells in the bone marrow with age, extending to all visceral organs. Most severe storage phenomena were found in the mononuclear-macrophage system, however, parenchymal cells of visceral organs were also markedly involved. The ultrastructural appearance of membrane-bound inclusions displayed a pleomorphic aspect ranging from small vesicular and vesiculo-granular structures to huge lysosomes with membranous material deposited in lamellar or stacked arrays. The obvious similarity to its human counterpart along with an easy availability makes this animal model a valuable tool for further studies of Niemann-Pick disease type A.