John Kilkus

Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli

An immortalized dorsal root ganglion cell line F‐11 exhibits many properties of spinal cord neurons and undergoes apoptosis in response to growth factor withdrawal and the exogenous addition of inhibitors of phosphatidylinositol‐3‐kinase (PI3K). To elucidate the mechanism of apoptosis we generated F‐11 clones which overexpressed either the p110 subunit of PI3K, a constitutively active form of protein kinase B/Akt (Myristoylated Akt), or a dominant‐negative form (c‐Akt). The first two constructs were protective against apoptosis induced by PI3K inhibitors such as wortmannin and LY294002. Caspase‐3 (CPP32) levels peaked at 4 hr to 6 hr in response to pro‐apoptotic drugs, and this increase was attenuated by 50% in F‐11 with constitutively active Akt. The Akt protection was confirmed by DNA fragmentation studies. Both neo‐transfected and the c‐Akt dominant‐negative transfected F‐11 cells showed increased ceramide formation (twofold) in response to staurosporine, wortmannin, or LY294002; whereas cells with a constitutively active Akt (Myr‐Akt) showed no increase in ceramide when treated with staurosporine, wortmannin, or LY294002. Ceramide was a more potent activator of CPP32 and an inducer of apoptosis when added as the native form (hydroxy‐ or nonhydroxy‐), rather than the more water‐soluble C2‐ceramide. Overexpression of PI3K (p110) and Akt protected cells against ceramide‐induced apoptosis, suggesting that Ceramide action is upstream of Akt in these cells and suggesting that Akt might be a target for inhibition by ceramide. Both staurosporine and C2‐ceramide activated the Jun kinase (JNK) cascade and C2‐ceramide increased caspase‐3 (CPP32) activity in cells expressing wild‐type c‐Jun, but not dominant‐negative (TAM‐67) c‐Jun. We suggest that this pathway is also involved in apoptosis, consistent with the idea that ceramide has multiple kinase and kinase‐modulating targets in the apoptotic pathway of neurons. J. Neurosci. Sci. 57:884–893, 1999.

Ceramide in rafts (detergent-insoluble fraction) mediates cell death in neurotumor cell lines

Detergent‐resistant lipid microdomains (Rafts) were isolated from human oligodendroglioma (HOG), human neuroblastoma (LA‐N‐5), and immortalized dorsal root ganglion (F‐11) cell lines by sucrose‐density gradient ultracentrifugation and shown to be enriched in cholesterol, sphingomyelin, and ceramide. [3H]palmitate labeling allowed the Raft fraction to be easily identified as a sharp peak of 3H radioactivity in the 5–30% sucrose interphase. Treatment of [3H]palmitate‐labeled cells with staurosporine (to activate caspase 8 and induce apoptosis) or exogenous sphingomyelinase specifically increased the [3H]ceramide content of the Raft fraction. Depletion of cholesterol with β‐methylcyclodextran decreased Raft formation and partially blocked staurosporine‐induced apoptosis. Similarly, treatment of cells with Fumonisin B1 to inhibit de novo sphingolipid synthesis by 50% reduced the labeling of the Raft fraction and partially blocked staurosporine‐induced apoptosis. Staurosporine treatment activated neutral sphingomyelinase but had no effect on acid sphingomyelinase activity or on other lysosomal hydrolases, such as α‐L‐fucosidase. Most of the neutral sphingomyelinase activity is in the Raft fraction, suggesting that the conversion of sphingomyelin to ceramide in Rafts is an important event in neural cell apoptosis.

Differential regulation of ceramide in lipid-rich microdomains (rafts): antagonistic role of palmitoyl:protein thioesterase and neutral sphingomyelinase 2.

Cell differentiation and myelination involve a fine balance between stasis and programmed cell death, yet the genes that regulate this have not been clearly defined. We therefore studied two key gene products involved in oligodendrocyte plasma membrane lipid metabolism and their antagonistic role in ceramide‐mediated cell death signaling. Overexpression of palmitoyl:protein thioesterase (PPT1; verified by Western blot of the V5‐tagged protein and increased enzyme activity) resulted in decreased ceramide in the detergent‐resistant microdomain (DRM, or raft) relative to cholesterol and sphingomyelin (SM). This PPT1 overexpression also resulted in protection against cell death induced by either staurosporine or C2‐ceramide. In contrast, overexpression of neutral sphingomyelinase 2 (NSMase2; verified by Western blot of the FLAG‐tagged protein and increased enzyme activity) resulted in increased membrane NSMase and increased ceramide in rafts relative to cholesterol and SM. The difference in SM and ceramide turnover was quantitated by [3H]palmitate pulse‐chase labeling. Furthermore, when NBD‐SM was added to cells, it was hydrolyzed by NSMase‐transfected cells at more than twofold the rate in untransfected cells. NSMase2 overexpression enhanced cell death induced by staurosporine or C2‐ceramide, in contrast to the protective effect of PPT1 overexpression. The presence of a fraction of both PPT1 and NSMase2 in rafts together with their substrates (palmitoylated proteins and SM, respectively) suggests a mechanism for dynamic palmitoylation/depalmitoylation of certain proteins in controlling cell death via NSMase activation.

ABSENCE OF OLIGODENDROGLIAL GLUCOSYLCERAMIDE SYNTHESIS DOES NOT RESULT IN CNS MYELIN ABNORMALITIES OR ALTER THE DYSMYELINATING PHENOTYPE OF CGT-DEFICIENT MICE

To examine the function of glycosphingolipids (GSLs) in oligodendrocytes, the myelinating cells of the central nervous system (CNS), mice were generated that lack oligodendroglial expression of UDP‐glucose ceramide glucosyltransferase (encoded by Ugcg). These mice (Ugcgflox/flox;Cnp/Cre) did not show any apparent clinical phenotype, their total brain and myelin extracts had normal GSL content, including ganglioside composition, and myelin abnormalities were not detected in their CNS. These data indicate that the elimination of gangliosides from oligodendrocytes is not detrimental to myelination. These mice were also used to asses the potential compensatory effect of hydroxyl fatty acid glucosylceramide (HFA‐GlcCer) accumulation in UDP‐galactose:ceramide galactosyltransferase (encoded by Cgt, also known as Ugt8a) deficient mice. At postnatal day 18, the phenotypic characteristics of the Ugcgflox/flox;Cnp/Cre;Cgt−/− mutants, including the degree of hypomyelination, were surprisingly similar to that of Cgt−/− mice, suggesting that the accumulation of HFA‐GlcCer in Cgt−/− mice does not modify their phenotype. These studies demonstrate that abundant, structurally intact myelin can form in the absence of glycolipids, which normally represent over 20% of the dry weight of myelin.

Ceramide regulation of the tumor suppressor phosphatase PTEN in rafts isolated from neurotumor cell lines.

The neutral sphingolipid ceramide has been implicated in the apoptotic death of cells by a number of different mechanisms, including activation of protein kinase B (Akt) phosphatase. Here we present evidence that ceramide recruits the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) into membrane microdomains (rafts), where it could act to reduce the levels of polyphosphoinositides necessary for the activation of Akt. A PTEN construct with a red‐fluorescent protein (RFP) tag was overexpressed in both a human cell line derived from oligodendroglioma (HOG) and a rat pheochromocytoma cell line (PC12) by means of an inducible promoter system (Tet‐Off). Induction of PTEN by removal of doxycycline enhanced both capsase‐3 and cell death with staurosporine, wortmannin, or C2‐ceramide, whereas antisense PTEN had the reverse effect. Overexpression of PTEN also increased acid sphingomyelinase (ASMase) activity. PTEN normally has a generalized (cytosolic/membrane) distribution, but treatment with C2‐ceramide translocated a fraction of the PTEN to the plasma membrane, showing a plasma membrane distribution similar to that observed for a prenylated green‐fluorescent (GFP) construct. PTEN was then shown to translocate to the detergent‐resistant membrane microdomain fraction (raft) of the plasma membrane. The colocalization of sphingomyelinases, ceramide, polyphosphoinositides, and PTEN in the raft fraction further suggests that the association of these lipids is critical for regulating cell death.

Mitochondrial abnormalities in CLN2 and CLN3 forms of batten disease

The storage of subunit c of mitochondrial ATP synthase, other hydrophobic peptides, and autofluorescent pigment in both late infantile (CLN2) and juvenile (CLN3) neuronal ceroid lipofuscinosis, but not in infantile (CLN1), has raised the question of abnormal mitochondrial function. We now report a partial deficiency in three types of fatty acid oxidation in intact skin fibroblasts from CLN2 and CLN3 patients, but not CLN1. We observed a statistically significant 33% reduction in palmitate (beta-oxidation; mainly mitochondrial) and lignocerate (beta-oxidation; mainly peroxisomal), and a 50% reduction in phytanic acid (alpha-oxidation; mainly peroxisomal) in the absence of exogenous carnitine. In contrast, when we measured fatty acid beta-oxidation (lignoceric acid and palmitic acid), in the same human skin fibroblasts, following lysis in the presence of carnitine, we found no difference in enzyme activity among normal, CLN1, CLN2, and CLN3. However, we did observe a 40% reduction in peroxisomal particulate (bound) catalase activity in CLN1 and CLN2 fibroblasts, which typically results from organellar lipid accumulation or a membrane abnormality. However, total catalase levels were normal, and Western blot analysis of this and three other major oxidant protective enzymes (Mn-dependent superoxide dismutase [MnSOD], CuZn-dependent superoxide dismutase [CuZnSOD], and glutathione peroxidase) were normal in CLN1, CLN2, and CLN3, as well as in liver from an animal (English Setter dog) model for CLN, which shows similar pathology and subunit c storage. Our data showing differences between CLN1 and forms CLN2 and CLN3 suggest some type of mitochondrial membrane abnormality as the source of the pathology in CLN2 and CLN3.

Oxidized phosphatidylcholine formation and action in oligodendrocytes.

Reactive oxygen species play a major role in neurodegeneration. Increasing concentrations of peroxide induce neural cell death through activation of pro‐apoptotic pathways. We now report that hydrogen peroxide generated sn‐2 oxidized phosphatidylcholine (OxPC) in neonatal rat oligodendrocytes and that synthetic OxPC [1‐palmitoyl‐2‐(5′‐oxo)valeryl‐sn‐glycero‐3 phosphorylcholine, POVPC] also induced apoptosis in neonatal rat oligodendrocytes. POVPC activated caspases 3 and 8, and neutral sphingomyelinase (NSMase) but not acid sphingomyelinase. Downstream pro‐apoptotic pathways activated by POVPC treatment included the Jun N‐terminal kinase proapoptotic cascade and the degradation of phospho‐Akt. Activation of NSMase occurred within 1 h, was blocked by inhibitors of caspase 8, increased mainly C18 and C24:1 ceramides, and appeared to be concentrated in detergent‐resistant microdomains (Rafts). We concluded that OxPC initially activated NSMase and converted sphingomyelin into ceramide to mediate a series of downstream pro‐apoptotic events in oligodendrocytes.

Differential regulation of sphingomyelin synthesis and catabolism in oligodendrocytes and neurons

Neurons (both primary cultures of 3‐day rat hippocampal neurons and embryonic chick neurons) rapidly converted exogenous NBD‐sphingomyelin (SM) to NBD‐Cer but only slowly converted NBD‐Cer to NBD‐SM. This was confirmed by demonstrating low in vitro sphingomyelin synthase (SMS) and high sphingomyelinase (SMase) activity in neurons. Similar results were observed in a human neuroblastoma cell line (LA‐N‐5). In contrast, primary cultures of 3‐day‐old rat oligodendrocytes only slowly converted NBD‐SM to NBD‐Cer but rapidly converted NBD‐Cer to NBD‐SM. This difference was confirmed by high in vitro SMS and low SMase activity in neonatal rat oligodendrocytes. Similar results were observed in a human oligodendroglioma cell line. Mass‐Spectrometric analyses confirmed that neurons had a low SM/Cer ratio of (1.5 : 1) whereas oligodendroglia had a high SM/Cer ratio (9 : 1). Differences were also confirmed by [3H]palmitate‐labeling of ceramide, which was higher in neurons compared with oligodendrocytes. Stable transfection of human oligodendroglioma cells with neutral SMase, which enhanced the conversion of NBD‐SM to NBD‐Cer and increased cell death, whereas transfection with SMS1 or SMS2 enhanced conversion of NBD‐Cer to NBD‐SM and was somewhat protective against cell death. Thus, SMS rather than SMases may be more important for sphingolipid homeostasis in oligodendrocytes, whereas the reverse may be true for neurons.

CrmA protects against apoptosis and ceramide formation in PC12 cells.

TNF-α activated caspase 8 and caspase 3 in PC12 cells, leading to cell death by apoptosis (DNA fragmentation). TNF-α caspase activation and cell killing were blocked by transfection and overexpression of the viral protein CrmA, which specifically inhibits caspase 8. CrmA was also able to block the TNF-α-induced increase in ceramide formation in PC12 cells. Conversely, if caspase 8 was activated by light-activated Rose Bengal, there was an increase in both ceramide and caspase 3–mediated apoptosis, which was blocked by CrmA overexpression. This suggested that caspase 8 increases ceramide either by increasing its synthesis or by activating sphingomyelinase. Since fumonisin B1 did not block and sphingomyelin decreased when ceramide increased, we concluded that activation of sphingomyelinase is the most likely mechanism. The Rose Bengal activation of caspase 8 and increased ceramide formation was blocked with IETD-CHO, to show that reactive oxygen species (also generated by Rose Bengal) were not responsible for the observed increase in ceramide. Thus in PC12 pheochromocytoma cells, ceramide appears to amplify the death signal and there appears to be a sequence of events: TNF; TRADD, pro-caspase 8, caspase 8, sphingomyelinase, ceramide, caspase 3, apoptosis.

Multiple sphingolipid abnormalities following cerebral microendothelial hypoxia

Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]‐palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long‐chain forms, was observed in both whole cells and detergent‐resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW‐4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro‐sphingosine (DH‐Sph), DH‐sphingosine1‐phosphate (DH‐S1P), DH‐sphingomyelin (DH‐SM), DH‐glucosylceramide (DH‐GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3‐mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydro‐sphingolipids, including DHC, DH‐SM, DH‐GlcCer, DH‐Sph and DH‐S1P, which may be involved in ameliorating the effects of stroke .