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Lysosphingolipids potently and reversibly inhibited protein kinase C activity and binding of phorbol dibutyrate in vitro and in human platelets. As with activation of protein kinase C by phosphatidylserine and sn-1,2-diacylglycerol, inhibition was subject to surface dilution. Accordingly, inhibition in mixed micelle assays was dependent on the molar percentage of lysosphingolipids rather than the bulk concentration. Lysosphingolipids inhibited protein kinase C activity at molar percentages similar to those required for activation by phosphatidylserine and sn-1,2-diacylglycerol. Since lysosphingolipids accumulate in Krabbe's disease, Gaucher's disease, and other sphingolipidoses, the hypothesis that lysosphingolipid inhibition of protein kinase C represents the missing functional link between the accumulation of sphingolipids and the pathogenesis of these disorders appears to unify existing data. The accumulation of lysosphingolipids would cause progressive dysfunction of signal transduction mechanisms vital for neural transmission, differentiation, development, and proliferation and would eventually lead to cell death.

3-Keto derivatives were prepared in good yield by the oxidative procedure with 2,3-dichloro-5,6-dicyanobenzoquinone from N -acetyl sphingosine, N -palmitoyl sphingosine, N -lignoceroyl sphingosine, and N -lignoceroyl psychosine. None of these 3-keto derivatives, except the one from N -acetyl sphingosine, have been previously reported. Ceramides were isolated from a calf brain and reacted with 2,3-dichloro-5, 6-dicyanobenzoquinone. Ceramides containing sphingosine (4-sphingenine) were converted to 3-keto derivative, while those containing dihydrosphingosine (sphinganine) remained intact under these conditions. The 3-keto ceramides were then separated from the ceramides containing dihydrosphingosine by preparative thin layer chromatography. Similarly cerebrosides from the same calf brain were oxidized and fractionated to 3-ketocerebrosides (from cerebrosides containing sphingosine) and unreacted cerebrosides (cerebrosides containing dihydrosphingosine). The fatty acid composition of these four sphingolipids were determined. Both the ceramides and the cerebrosides containing sphingosine had more unsaturated fatty acids than the corresponding dihydrosphingosine-containing compounds. The ratio of C 16 -C 20 fatty acids to C 22 -C 26 acids was higher in the ceramides containing sphingosine than in ceramides containing dihydrosphingosine, while the ratio was reversed in cerebrosides. The possible precursor-product relationship among these lipids is discussed.

The sera of 71 patients with colorectal cancer were examined for lactosylsphingosine-reactive antibody and CEA. Fifteen of those patients were studied repeatedly over extensive periods of time. The antibody was determined by the semiquantitative radioimmunoabsorption technique using lactosylsphingosine-polyacrylamide conjugate and [ 125 I ]-labelled anti-human IgG. Excessive antibody levels were invariably found in serum samples of all 39 patients who were examined before or within 2 months after surgery. Serum samples of certain patients became negative for the presence of high antibody levels usually between 3 and 6 months after surgery. This occurred in 13 out of 41 operated patients. The follow-up study revealed that 11 such patients have been free of any signs of cancer relapse up until the time of the follow-up examination, i.e. for 12–28 months , mean 19 months . In contrast, only 4 out of 28 patients who remained positive for the excessive antibody longer than 2 months after surgery are at present free of the disease. The high antibody levels which persist more than 6 months after surgery are almost always associated with cancer recurrences or metastases. This was true for 21 out of 22 such patients. The high levels of the antibody preceded other signs of cancer relapse, including increased concentrations of CEA in about 40% of the operated patients.

The possible involvement of protein kinase C on luteinizing hormone (LH) effects in dispersed rat luteal cells was investigated using two substances that have been reported to be protein kinase C inhibitors, sphingosine and psychosine. Sphingosine efficiently inhibited protein kinase C activity both in brain and luteal cytosol fractions. Both substances inhibited LH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in a dose-dependent fashion with an ID 50 at 3–7 μM (sphingosine) and 40 μ.m (psychosine). LH-stimulated progesterone production was also inhibited with an ID 50 at 6–10 μM (sphingosine) and 40–100 μM (psychosine). The inhibition was not due to an increased phosphodiesterase activity since IBMX (3-isobutyl-1-methylxanthine, 0.1 mM) and RO 20–1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, 0.1 mM) did not abolish the inhibitory effect of sphingosine. To study the mode of action of sphingosine, forskolin and cAMP analogues were tested. The effect of these substances on Steroidogenesis was inhibited, as well as the forskolin-induced cAMP accumulation, by sphingosine. This study demonstrates a clear inhibition of LH-stimulated effects by sphingosine and psychosine. LH action in rat luteal cells is discussed in relation to protein kinase C and the possible mode of sphingosine action.

A sensitive and specific analytical procedure has been developed for determination of galactosylsphingosine (psychosine) in the brain. The method takes advantage of two unusual properties of psychosine--the strong positive charge and the reactivity of the free amino group. It involves lipid extraction, separation from other lipids on a cation-exchange column (AG-50W), elimination of the last trace of galactosylceramide by silicic acid chromatography, dansylation of psychosine, Florisil and DEAE-Sephadex chromatography, and finally, fluorescent densitometry of dansylated psychosine separated by thin-layer chromatography. The detection limit is 5-10 ng/100 mg brain tissue when the standard procedure is followed exactly. Reliable determination can be made for 50 ng/100 mg or higher in the presence of 200,000-fold excess of other lipids and in the presence of 40,000-fold excess of galactosylceramide. The sensitivity can be increased fivefold by using a larger aliquot for the final determination. This analytical procedure has been successfully applied to demonstrate abnormal accumulation of psychosine in the brain in human, canine, and murine genetic galactosylceramidase deficiencies (globoid cell leukodystrophy).

Leukodystrophies are disorders affecting primarily oligodendroglial cells or myelin. Another necessary criteria for defining a leukodystrophy is that the disorder has to be of endogenous origin with a pattern compatible with genetic transfer of a metabolic defect. The clinical criterion of a steadily progressive deterioration of function must also be included. Much of the material presented during the conference related to three leukodystrophies from which subclasses with a relatively uniform clinical presentation can be distinguished and about which a considerable body of consistent biochemical information is available. These disorders are metachromatic leukodystrophy (MLD), globoid leukodystrophy (GLD, also referred to as Krabbe's disease) and adrenoleukodystrophy (ALD). Discussion was focused to the pathophysiology of the more prevalent "classical" subclass of each of these disorders; discussion of clinical variants was in the context of what we could learn about the more prevalent form. Finally, because of time restraints, most of the discussion on the final day was centered around MLD. This clinical disorder was used as a starting point for discussion of questions both specific to MLD as well as those questions common to all leukodystrophies. The discussion of GLD and ALD was more restricted and only points relevant to those specific leukodystrophies were discussed. Information from the presentation not dealing directly with these human disorders is also summarized.

We have previously shown that cyclic AMP (cAMP) inhibits the protein kinase C (PKC)-mediated phosphorylation of myelin basic protein (MBP) in cultured oligodendrocytes (OLGs). Recently, it has been demonstrated that the long chain base sphingosine inhibits PKC by competing PKC effectors (diacylglycerol and phorbol esters) for a binding site on the kinase (Hannun and Bell: Science 235: 670-674, 1987). In this report we define further the mechanism by which cAMP inhibits MBP phosphorylation by comparing the effects of cAMP with that of galactosylsphingosine (psychosine), a potential catabolite of galactocerebroside, the major OLG glycosphingolipid. We identify the consequences of psychosine treatment and PKC down-regulation on OLG morphology and electrophysiology and discuss their relevance. Our results in intact ovine oligodendrocytes are consistent with a mechanism in which cAMP inhibits MBP phosphorylation by interfering with the release of diacylglycerol (DAG) from phosphatidylinositol. First, the effects of cAMP on MBP phosphorylation are reversed with exogenous TPA; and second, cAMP inhibits the incorporation of 1-[14C]arachidonate into DAG and specifically inhibits the turnover (as judged by 32PO4 3-incorporation) of phosphatidylinositol. Psychosine inhibits MBP phosphorylation, and its action can be reversed by TPA suggesting a mechanism of inhibition similar to that described for other systems. In addition, psychosine has profound effects on OLG morphology; it disintegrates OLG processes while leaving the cell soma intact. Stable hyperpolarized resting potentials were obtained following psychosine treatment, but there was a 66% decrease in membrane capacitance indicating a significant decrement in membrane surface area. The morphological changes induced by psychosine are reversible and can be eliminated by removing the drug but not by the addition of TPA. Whether inhibition of PKC by psychosine plays any role in process dissolution remains an unanswered question. However, current evidence suggests that a PKC-independent mechanism may be at play. This investigation in conjunction with our previous work emphasizes a role for the interregulation of protein kinase A (PKA) and PKC in the control of OLG somal vs. myelin components. This may have significant implications for central nervous system myelin assembly.

Neuronal cell culture system has been used for the study of pathochemical evaluations in human degenerative brain disorders, particularly for Krabbe's disease and neuronal ceroid lipofuscinosis. To understand the pathochemistry of Krabbe's disease, we added psychosine into neuronal cell cultures and psychosine treated cells showed the destruction of cytoskeleton and pathy intracellular changes. Electron microscopic finding showed the swelling of the mitochondria. Oligodendrocytes and Schwann cells were isolated from the brains and sciatic nerve of twitcher mouse as an authentic murine model of globoid cell leukodystrophy. Oligodendroglial cells cultured for 22 days were stained by anti-galactocerebroside antibodies. In twitcher oligodendrocyte processes were wirelike and progressively degenerated and there were few membranous expansion. Schwann cells from twitcher could not elongated their processes. These data suggest that psychosine might be important factor to result in these pathological conditions. Furthermore, we studied the effect of protease inhibitors, E-64 on dissociated primary cultures from fetal rat brain. After treated with E-64 in a concentration from 0.1-50 micrograms/ml, numerous cytoplasmic accumulations appeared in neuronal cells. These morphological pictures resemble with those of neuronal ceroid lipofuscinosis, Batten disease. We will discuss the relationship between the deficiency of catepsin H in Batten disease and inclusion bodies found in E-64.

A method of thin-layer chromatography was used for the separation of glycolipids from lipid extracts of animal tissues. The technique of two-step development was applied. In the first step glycolipids are separated from phospholipids; the second step makes possible the separation of different glycolipid classes, viz . ceramide mono-, tri- and tetrahexosides, sulphatides and monogalactosyl diglycerides. Gluco- and galactolipids were separated on plates partially impregnated with sodium boarate. The separation of psychosine, sphingosine and ceramide is also described.

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [3H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intra-nucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40–80 μM of lysosphingolipids for 24–48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37 °C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation. These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses. —Sueyoshi, N., T. Maehara, and M. Ito. Apoptosis of Neuro2a cells induced by lysosphingolipids with naturally occurring stereochemical configurations.

Monoclonal antibody to Geodia lectin is bound by the mucocyst content of Tetrahymena. By using this (fluorescent-labelled) antibody and confocal microscopy, the actual state of the mucocyst (position, resting, filling or extruding) can be studied. Treatment with C2 ceramide and non-hydroxy fatty acid caused a rapid depletion of mucocyst material. Another ceramide analogue, psychosine caused fusion of mucocysts. In these cases--in contrast to the controls--the contractile vacuole was filled with mucocyst material and this was seen in the tubules in contact with the contractile vacuoles. Hydroxy fatty acid ceramide, sphingomyelin and sphingosine-1-phosphate were ineffective. As the former materials influence also the cytoskeleton, while the latter do not, the cytoskeleton is presumed to have a mediatory effect. Neither the connection of contractile vacuoles with tubular structures nor mucocyst fusion have been described before.

Glucosylceramide, a degradation product of complex glycosphingolipids, is hydrolysed in lysosomes by glucocerebrosidase (GlcCerase). Mutations in the human GlcCerase gene cause a reduction in GlcCerase activity and accumulation of glucosylceramide, which results in the onset of Gaucher disease, the most common lysosomal storage disease. Significant clinical heterogeneity is observed in Gaucher disease, with three main types known, but no clear correlation has been reported between the different types and levels of residual GlcCerase activity. We now demonstrate that a correlation exists by using a radioactive, short-acyl chain substrate, N-(1-[14C]hexanoyl)-D-erythro-glucosylsphingosine ([14C]hexanoyl-GlcCer). This substrate rapidly transferred into biological membranes in the absence of detergent [Futerman and Pagano (1991) Biochem. J. 280, 295-302] and was hydrolyzed to N-(1-[14C]hexanoyl)-D-erythro-sphingosine ([14C]hexanoyl-Cer) both in vitro and in situ, with an acid pH optimum. A strict correlation was observed between levels of [14C]hexanoyl-GlcCer hydrolysis and Gaucher type in human skin fibroblasts. The mean residual activity measured in vitro for 3 h incubation in type 1 Gaucher fibroblasts (the mild form of the disease) was 46.3 +/- 4.6 nmol of [14C]hexanoyl-Cer formed per mg protein (n = 9), and in type 2 and 3 fibroblasts (the neuronopathic forms of the disease) was 19.6 +/- 6.5 (n = 9). A similar correlation was observed when activity was measured in situ, suggesting that the clinical severity of a lysosomal storage disease is related to levels of residual enzyme activity.

Globoid leukodystrophy (Krabbe's disease) was diagnosed in an infant in whom a progressive neurological disorder and optic atrophy developed. At autopsy, ultrastructural examination of the optic nerves and cerebral white matter revealed characteristic tubular inclusions in globoid-epithelioid cells. Thinning of the nerve fiber and ganglion cell layers of the retina appeared to be due to retrograde degeneration of the optic nerve related to the abnormal metabolism of myelin. We discuss the pathogenesis of the clinical and pathological ocular findings, with regard to the inherited absence of the enzyme galactocerebroside beta-galactosidase and the accumulation in the optic nerve and brain of its substrates, galactocerebroside and psychosine.

Lysosomal storage diseases are a group of disorders where accumulation of catabolites is manifested in the lysosomes of different cell types. In metachromatic leukodystrophy (Arylsulfatase A [EC.3.1.6.8] deficiency) storage of the glycosphingolipid sulfatide in the brain leads to demyelination, resulting in neuromotor co-ordination deficits and regression. In a mouse model for metachromatic leukodystrophy, the ASA null mutant mouse, the accumulation of sulfatide in correlation to phenotype has been thoroughly investigated. Another lipid species reported to accumulate in patients with metachromatic leukodystrophy is the sulfatide related lipid lysosulfatide. Lysosulfatide was shown to be a cytotoxic compound in cell culture experiments and thus suggested to be involved in the pathology of metachromatic leukodystrophy. In this study, we further investigated the developmental profile of lysosulfatide in the brain of ASA null mutant mice by using high performance liquid chromatography. Lysosulfatide could be detected in the brain of normal mice (ASA +/+) from 1.8 months up to 23.1 months of age. From the age of 8.8 months the lysosulfatide levels remained constant at 1 pmol/mg wet tissue. The developmental change (< 20 months) of brain lysosulfatide showed an accumulation in ASA null mutant mice at ages above one month compared to its normal counterpart (ASA +/+). Thus, the ASA null mutant mouse might be a suitable model to further investigate the role of lysosulfatide in the pathogenesis of metachromatic leukodystrophy.