Regulation of sphingomyelinases in cells of the oligodendrocyte lineage

Controversy exists regarding the nature of the "executioner" sphingomyelinase (SMase) in cells and its subcellular localization. A new fluorescence-based assay with the substrate 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine allowed rapid and reliable microassays of neutral (N) and acid (A) SMase activity in cell extracts from primary cultures of neonatal rat oligodendrocytes (OPC) and a human oligodendroglioma cell line (HOG). Total SMase activity was much higher in OPC than in HOG cells. Both staurosporine and tumor necrosis factor-alpha (TNF-alpha) induced apoptosis and activated NSMase in a multiphasic manner in both OPC and HOG cells. The increase in caspase 8 activity preceded the 1 hr peak of NSMase activation, which was followed by caspase 3 activation. In contrast, ASMase activity, which constituted >90% of the total SMase activity, was unresponsive to proapoptotic drugs. Neither reducing ASMase levels by 50% by pretreatment with desipramine nor inhibiting sphingolipid synthesis by 50% with fumonisin B1 had any effect on cell death. Isolation of sphingolipid-rich plasma membrane microdomains (rafts) from the cells by sucrose density gradient ultracentrifugation revealed an enrichment of sphingomyelin, ceramide, and caspase 8. Proapoptotic drugs such as staurosporine promoted the translocation of NSMase to the raft fraction. In contrast, ASMase, other lysosomal hydrolases, and caspase 3 remained absent from rafts even after staurosporine treatment. The staurosporine-induced concomitant increase of ceramide in the raft fraction and caspase 3 in the cytosol could be mimicked by the addition of exogenous bacterial SMase. We conclude that caspase 8 activates NSMase in rafts in oligodendrocytes and that the downstream apoptotic signal is via caspase 3.     

Antisense palmitoyl protein thioesterase 1 (PPT1) treatment inhibits PPT1 activity and increases cell death in LA-N-5 neuroblastoma cells

Infantile neuronal ceroid lipofuscinosis (INCL) is a childhood neurodegenerative disease caused by the selective death of cortical neurons and retinal degeneration, as the result of a palmitoyl protein thioesterase 1 (PPT1) deficiency. Recently, we showed that overexpression of PPT1 protects LA-N-5 human neuroblastoma cells against apoptotic death (Cho and Dawson [2000a] J. Neurochem. 74:1478-1488) and we now show that inhibition of PPT1 increases the susceptibility of these cells to apoptotic cell death. Transient transfection of LA-N-5 neuroblastoma cells with PPT1-FLAG resulted in a strong expression of PPT-FLAG-tagged protein as evidenced by Western blot analysis and immunofluorescence. Co-transfection of a reverse-oriented (antisense) PPT1 (AS-PPT1) decreased the expression of PPT-FLAG to almost zero, reduced PPT1 enzyme activity (as measured by an in vitro assay) and increased the susceptibility to apoptosis induced by C(2) ceramide. Similarly, inhibition of PPT1 with a synthetic inhibitor (AcG-palmitoyl diaminoproprionate-VKIKK) (DAP1) (100 microM) increased the susceptibility of the cells to apoptosis induced by either C(2)-ceramide or etoposide, a common chemotherapeutic agent used in the treatment of neuroblastoma. Cells stably overexpressing PPT1 were resistant to apoptosis induced by DAP1 suggesting that the inhibitor has a specific action and confirming that low levels of protein palmitoylation block the death pathway. Drugs that raise the level of protein palmitoylation are pro-apoptotic and PPT1 inhibition may enhance the killing efficacy of chemotherapeutic agents used to kill neuroblastoma-derived cells.     

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.     

Increased ceramide in brains with Alzheimer's and other neurodegenerative diseases

Ceramide has been suggested to participate in the neuronal cell death that leads to Alzheimer's disease (AD), but its role is not yet well-understood. We compared the levels of six ceramide subspecies, which differ in the length of their fatty acid moieties, in brains from patients who suffered from AD, other neuropathological disorders, or both. We found elevated levels of Cer16, Cer18, Cer20, and Cer24 in brains from patients with any of the tested neural defects. Moreover, ceramide levels were highest in patients with more than one neuropathologic abnormality. Interestingly, the range of values was higher among brains with neural defects than in controls, suggesting that the regulation of ceramide synthesis is normally under tight control, and that this tight control may be lost during neurodegeneration. These changes, however, did not alter the ratio between the tested ceramide species. To explore the mechanisms underlying this dysregulation, we evaluated the expression of four genes connected to ceramide metabolism: ASMase, NSMase 2, GALC, and UGCG. The patterns of gene expression were complex, but overall, ASMase, NSMase 2, and GALC were upregulated in specimens from patients with neuropathologic abnormalities in comparison with age-matched controls. Such findings suggest these genes as attractive candidates both for diagnostic purposes and for intervening in neurodegenerative processes.     

Differential effects of Bcl-2 overexpression on fibre outgrowth and survival of embryonic dopaminergic neurons in intracerebral transplants

The causes of death of transplanted neurons are not known in detail, but apoptotic mechanisms involving caspase activation are likely to play a role. We examined whether overexpression of the anti-apoptotic protein Bcl-2 may enhance the survival of dopaminergic [tyrosine hydroxylase (TH)-immunoreactive] grafted neurons. For this purpose, we prepared cells from embryonic day 13 ventral mesencephalon (VM) of mice overexpressing human Bcl-2, or from their wild-type littermates. The bcl-2 transgene was strongly expressed in these cells, and resulted in protection of neuronal cultures from death triggered by serum deprivation or exposure to staurosporine. To model pretransplantation stress more closely in vitro, we stored dissociated embryonic mesencephalic cells for 8 h in the same type of medium used for intracerebral transplantation. This resulted in massive cell death as quantified by lactate dehydrogenase (LDH) release, and increased DNA fragmentation. Although this cell loss was strongly reduced by a caspase inhibitor, Bcl-2 had no significant protective effect. Finally, mesencephalic cell suspensions were xenografted into the striatum of immunosuppressed hemiparkinsonian rats. Neither the survival of TH-immunopositive transplanted neurons nor the functional recovery of the rats was improved by Bcl-2, although the Bcl-2 protein was strongly expressed in transgenic grafts 5 weeks after implantation, and dopaminergic fibre outgrowth from the grafts was significantly improved. These data suggest that cell death in neuronal transplants involves apoptotic mechanisms that can bypass negative regulation by Bcl-2.     

Sigma-1 receptors potentiate epidermal growth factor signaling towards neuritogenesis in PC12 cells: potential relation to lipid raft reconstitution

We previously demonstrated that overexpression of sigma-1 receptors (sigma-1R) potentiated neurite sprouting caused by nerve growth factor in PC12 cells (Takebayashi et al. 2002 J Pharmacol Exp Ther 202:1227-1237). In this study we examined if sigma-1R may be involved in the action of epidermal growth factor (EGF). EGF is conventionally recognized as a mitogenic factor that stimulates only the proliferation of various types of cells, including PC12 cells. We found here that in sigma-1 receptor-overexpressing PC12 cells (sigma-1R OE cells), EGF markedly stimulates neuritogenesis without affecting cellular proliferation. EGF receptors (EGFR) are largely reduced in lipid rafts and are enriched in non-raft regions in sigma-1R OE cells. The enrichment of EGFR in the non-raft region is correlated with enhanced downstream signaling of EGFR including the phosphorylation of both EGFR and extracellular signal-regulated kinases (ERKs). Destruction of cholesterol-containing rafts by treating cells with methyl-beta-cyclodextrin also causes a reduction of EGFR in lipid rafts, a concomitant increase in the phosphorylation of both EGFR and ERK, and an increase in the EGF-induced neurite sprouting in wildtype cells. Furthermore, while overexpression of sigma-1R increases the level of lipid raft-associated cholesterol, the overexpression alters the levels of gangliosides in lipid rafts: GM1 and GM2 are decreased, whereas GD1a is increased. We conclude that sigma-1R cause the remodeling of lipid rafts, at least by increasing the level of lipid raft-associated cholesterol and by altering the levels of certain critical lipid raft-forming gangliosides. sigma-1R may thus play an important role in directing EGF signaling towards neuritogenesis, perhaps by shifting EGFR from the lipid raft into non-raft regions.     

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. [(3)H]palmitate labeling allowed the Raft fraction to be easily identified as a sharp peak of (3)H radioactivity in the 5-30% sucrose interphase. Treatment of [(3)H]palmitate-labeled cells with staurosporine (to activate caspase 8 and induce apoptosis) or exogenous sphingomyelinase specifically increased the [(3)H]ceramide content of the Raft fraction. Depletion of cholesterol with beta-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 alpha-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.     

Perturbation of sphingolipid metabolism and ceramide production in HIV-dementia

Infection by the human immunodeficiency virus type 1 (HIV-1) often results in neurological dysfunction including HIV dementia (HIVD). Alterations in cytokine and redox balance are thought to play important roles in the pathogenesis of HIVD, but the specific mechanisms underlying neuronal dysfunction and death are unknown. Activation of cytokine receptors and oxidative stress can induce the production of ceramide from membrane sphingomyelin, and recent findings suggest that ceramide is an important mediator of a form of programmed cell death called apoptosis. We now report that levels of ceramide, sphingomyelin, and hydroxynonenal (HNE) are significantly increased in brain tissues and cerebrospinal fluid of HIVD patients. Exposure of cultured neurons to the neurotoxic HIV proteins gp120 and Tat resulted in increased cellular levels of sphingomyelin, ceramide, and HNE. The ceramide precursor palmitoyl-CoA sensitized neurons to Tat and gp120 toxicity, whereas an inhibitor of ceramide production reduced Tat and gp120-induced increases of ceramide and HNE and protected the neurons from Tat and gp120-induced death. These results suggest that HIV-1 infection may promote a lipid imbalance in neural cells, resulting in an overproduction of ceramide and consequent cellular dysfunction and death.     

Targeted expression of BCL-2 attenuates MPP+ but not 6-OHDA induced cell death in dopaminergic neurons

Neurodegenerative diseases such as Parkinson's disease exhibit complex features of cell death reflecting both the primary lesion as well as surrounding interconnected events. Because Bcl-2 family members are intimately involved in cell death processes, the present study used dopaminergic cultures from control, Bcl-2-overexpressing, or Bax-deficient genetically modified animals to determine the in situ effects of parkinsonism-inducing toxins. MPP(+)-mediated cell death was attenuated by Bcl-2 but did not require Bax. Accordingly, mutations or deletions within Bax heterodimerization domains, BH1, BH2, or BH3 had no effect on Bcl-2's ability to prevent cell death, whereas the cell-death suppressing BH4 domain did. Although both staurosporine and 6-OHDA induced apoptosis, overexpression of Bcl-2 only rescued cells from programmed cell death induced by staurosporine. Thus, differential cell death pathways are associated with these cytotoxic signals in primary models of Parkinson's disease.     

Mechanisms of apoptosis in embryonic cortical neurons (E6 and E7) in culture involve lipid signalling, protein phosphorylation and caspase activation

Cultured embryonic (E7) chick neurons, derived from cerebral hemispheres, underwent apoptosis in response to inhibitors of protein kinase C (staurosporine) and phosphatidylinositol-3-kinase (wortmannin and LY294002), in a dose- and time-dependent manner. This was monitored by loss of cell viability, increased DNA fragmentation, and activation of caspase-3-like activity, all of which were partially reversed by elevating the level of cAMP in the cells with Bt(2)cAMP or (Sp)cAMPS. Further studies revealed that an early step in apoptosis was the formation of ceramide from sphingomyelin, resulting from the activation of a neutral pH sphingomyelinase activity. Thus inhibitors of protein kinase C and phosphatidylinositol-3-kinase increased ceramide levels in the same time-frame as caspase-3 activation and DNA fragmentation. Neurons could also be killed by the addition of either water-soluble C2-ceramide (30 microM) or natural C22/24 ceramide (0.5 microM). In contrast to the apparent protective effect of ser/thr protein phosphorylation, a pro-apoptotic role for tyrosine phosphate phosphorylation was suggested by the ability of protein tyrosine phosphate phosphatase inhibitor, Bis(maltolato)oxovanadium (IV) (BMOV), to induce apoptosis in E7CH neurons. Thus BMOV (25 microM) killed 50% of E7CH neurons and B lymphocytes but not glial cells, or T-lymphocytes, suggesting the existence of a common apoptotic pathway in neurons and B-cells.We conclude that the major pathway for programmed cell death in embryonic chick neurons has many elements in common with that described for other cells but that there may be some unique aspects which can be used to protect embryonic neurons from opioid and other drug-enhanced apoptosis.     

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 C(2)-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 C(2)-ceramide activated the Jun kinase (JNK) cascade and C(2)-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.     

Involvement of de novo ceramide biosynthesis in lymphotoxin-induced oligodendrocyte death.

Both experimental and clinical studies suggest that lymphotoxin (LT) plays an important role in multiple sclerosis (MS) by inducing oligodendrocyte (OL) depletion. However, the mechanism of LT cytotoxicity is unknown. Because of the role of ceramide as a cell death mediator for a large variety of cytotoxic molecules, we have investigated the possible role of this second messenger in LT-induced cytotoxicity on SV40 immortalized new-born mice OL. Human recombinant LT exposure (50 ng/ml) resulted in intracellular ceramide accumulation which peaked at 48 h (approximately 170% increase) and paralleled LT-induced cytotoxicity. Moreover, fumonisin B1, a potent and specific ceramide synthase inhibitor, not only inhibited ceramide accumulation but also protected OL from LT cytotoxicity. These results suggest that LT-induced ceramide synthase stimulation and subsequent increased intracellular ceramide concentration are implicated in oligodendrocyte death.     

RETRACTED ARTICLE: Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia

Objective

To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5 (RGC-5) cells, pro-protein convertase-2 (PC2), carboxypeptidase-E (CPE) and preproneuropeptide Y (preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed.

Methods

The RGC-5 cell was differentiated in 0.1 μmol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation (OGD). The acute or chronic OGD-induced cell death rates were obtained by using PI or TUNEL staining. The protein expression levels were determined by using the Western blot method and PC2 activity analysis.

Results

The ischemia caused substantial cell death in an OGD dose-dependent manner. In the cells, proPC2 and preproNPY protein levels gradually increased whereas proCPE gradually decreased. After OGD, PC2 activity was decreased. In the conditioned medium, proPC2 and PC2 proteins gradually decreased whereas proCPE, CPE, and preproNPY proteins gradually increased.

Conclusion

These results demonstrated that OGD inhibited the neuropeptide pro-protein processing system by reducing PC2 activity and the maturation of proPC2. The aggregation of the pro-proteins and the increase of the active CPE excision adversely exacerbated the cell injury. The pro-protein processing system might play a critical role in the ischemic stress of RGC-5 cells.     

BACE1 interacts with lipid raft proteins

A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques in the brain. Amyloid-beta peptide (Abeta) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Abeta generation. In support of this, APP, Abeta, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Abeta are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT-1 binds to BACE1 (beta-site APP cleaving enzyme 1) and that overexpression of CAV-1 or FLOT-1 results in recruiting BACE1 into lipid rafts and influence on beta-secretase activity in cultured cells. Our results show that both CAV-1 and FLOT-1 may modulate beta-secretase activity by interacting with BACE1.     

Atypical PKC zeta is activated by ceramide, resulting in coactivation of NF-kappaB/JNK kinase and cell survival.

Both protein kinase C (PKC) and ceramide play a critical role in cell signaling, but the relationship between PKC and ceramide is unclear. Low concentrations of ceramide were observed to transiently stimulate PKC zeta activity in vitro and in vivo, whereas high doses of ceramide lead to inhibition of PKC zeta. Inhibition of activity was accompanied by enhanced binding of the negative regulator, Par4 to PKC zeta. Treatment of PC12 cells with low doses of ceramide promoted survival in serum-free media and activation of nuclear factor-KB, whereas higher doses (>2.5 microM) resulted in cell death. Overexpression of either aPKC isoform, PKC zeta or iota, resulted in enhanced survival of PC12 cells at high doses of ceramide and in ceramide-stimulated Jun N-terminal kinase (JNK), without any apparent effect on mitogen-activated kinase. These findings support a role for ceramide-induced PKC zeta activity in the control of cell survival signaling via a pathway that also activates JNK kinase.     

Palmitoyl protein thioesterase 1 is targeted to the axons in neurons

Palmitoyl protein thioesterase 1 (PPT1) is a depalmitoylating enzyme whose deficiency leads to infantile neuronal ceroid lipofuscinosis. The disease is characterized by early loss of vision and massive neuronal death. Although PPT1 is expressed in many tissues, a deficiency of PPT1 damages neurons only in the cerebral and cerebellar cortexes and retina; other cell types remain relatively unaffected. We previously demonstrated that PPT1 is present in the synaptosomes and synaptic vesicles of neurons. To understand the crucial role of PPT1 for neuronal cells, we further investigated the expression and targeting of PPT1 in retinal, hippocampal, and cortical neurons during their maturation in culture. We found that PPT1 activity increases by neuronal maturation and is highest in retinal neuron cultures. In retinal neurons the expression of PPT1 precedes that of the synaptic vesicle protein 2 and synaptophysin, indicating a significant role for PPT1 in the early development of neuronal cells. We also found by quantitative confocal immunofluorescence microscopy that PPT1 is targeted preferably to axons in mature neurons, as indicated by its colocalization with the axonal marker microtubule-associated protein 1. In axons PPT1 is targeted specifically to axonal varicosities and presynaptic terminals, as indicated by its significant colocalization with growth-associated protein 43 and synaptophysin. Axonal localization of PPT1 was confirmed by double labeling with synaptophysin and postembedding immunoelectron microscopy. The polarized axonal targeting of PPT1 may well indicate a role for PPT1 in the exocytotic pathway of neurons.     

Effect of Ginkgo biloba (EGb 761) on staurosporine-induced neuronal death and caspase activity in cortical cultured neurons

Previous studies suggest the protective potentiality of Ginkgo biloba (EGb 761) against apoptotic cell death induced by hydroxyl radicals, staurosporine, serum deprivation and beta-amyloid (betaA) peptide. We have extended these observations to cultured cortical neurons and studied the effect of EGb 761 on neuronal survival (evaluated as MTT reduction), the presence of condensed nuclei (monitored as Hoechst staining), the time-course of caspase-1, caspase-3 and caspase-9 activation (measured by cleavage of specific fluorescent substrates) and superoxide anion production (evaluated by hydroethidine staining) after the exposure to staurosporine. Results show that 200 microg/ml of EGb 761 increased cell survival and reduced the number of condensed nuclei after the exposure to 200 nM staurosporine. Vitamin E and the spin trapper alpha-phenyl-N-tert-butylnitrone (PBN) also significantly increased cell survival. In contrast, the broad-spectrum caspase inhibitors ZVAD and ZBIOT showed no protection. Similarly, selective inhibitors of caspase-1 (YVAD-CHO), caspase-2 (VDVAD-CHO), caspase-3 (DEVD-CHO) and caspase-8 (IETD-CHO) did not protect against cell damage induced by staurosporine. The protective effect of EGb 761 was not enhanced when coincubated with vitamin E or DEVD-CHO. Caspase-3 activity was maximally induced 5-8 h after staurosporine exposure. Both EGb 761 and vitamin E showed a tendency to decrease caspase-3 activity. In contrast, activation of caspase-1 and caspase-9 was not observed at any of the times studied after STS exposure. Exposure to staurosporine resulted in increased superoxide production that was maximal at 5 h. EGb 761 significantly inhibited superoxide production at short times after staurosporine exposure. Vitamin E and PBN also significantly reduced superoxide production. Results suggest that EGb 761 neuroprotective effect might be mediated by its well-known antioxidant activity, which might also influence caspase-3 activation. Inhibition of capase-3 induced by EGb 761 and vitamin E does not seem to contribute to their observed protective action.     

Neurovisceral lipidosis compatible with Niemann-Pick disease type C: Morphological and biochemical studies of a late infantile case and enzyme and lipid assays in a prenatal case of the same family

Summary One postnatal and one prenatal case (same family) of a neurovisceral lipidosis compatible with a diagnosis of Niemann-Pick disease type C were studied. The postnatal case, aged 4 and 6/12 at death, was characterized morphologically (foamy cells in the bone marrow; storage histiocytes in rectal submucosa and extraneural viscera and ballooned neurons, the two types of cells containing pleomorphic and oligomenbranous inclusion bodies, respectively; central demyelination) as well as biochemically (elevated spleen and liver content of sphingomyelin, cholesterol, glucosyl ceramide and lysobisphosphatidic acid). Sphingomyelinase activity (SM) was not significantly lowered and showed no greatly abnormal electrofocused pattern of activity; its extractability from brain, liver and spleen was distinctly hindered, a finding interpreted as expression of a reduced bioavailability of the enzyme. — The prenatal case was diagnosed by low SM in amniotic fluid. Diminished SM was confirmed in cultured amniotic cells and in tissues of the aborted fetus which, additionally, showed an elevated sphingomyelin and cholesterol content in the liver. A prenatal diagnosis of Niemann-Pick disease type C was made for the first time. The phenotypical variation of the disease may reflect genetic heterogeneity and, there-fore, a prenatally lowered SM need not be a constant finding. — The apparent normalization of SM in the postnatal case was accompanied by a decrease of visceromegaly raising the question of a causal relationship between the two phenomena.