Ultrastructural investigations on two lymphoid cell lines from Niemann-Pick disease type B

Lymphoid cell lines (LCL) were established by Epstein-Barr Virus (EBV) transformation of blood B-lymphocytes from two different patients affected with Niemann-Pick disease (NPD) type B. Those lines were severely deficient in sphingomyelinase activity (8% and 10% residual activity). Ultrastructural investigations showed in both these lines the presence of numerous osmiophilic, dense and pleiomorphic inclusions characteristic of lysosomal storage (due to the accumulation of amphiphilic lipids) similar to those observed in tissues from NPD.     

Epstein-Barr virus transformed lymphoid cell lines as a new model system in culture for the study of GM2-gangliosidoses: Tay-Sachs and Sandhoff diseases

Epstein-Barr Virus transformed cell lines (LCL) were established from blood B-lymphocytes of patients affected with GM2-gangliosidoses variant O (Sandhoff disease, SD) and variant B (Tay-Sachs disease, TSD). LCL from SD showed a severe deficiency of activity of the major lysosomal beta-N-acetylhexosaminidase isoenzymes, Hex A and B; the residual activity was due to Hex S and Hex C. In LCL from TSD, the whole Hex activity was not deficient but isoenzyme composition was completely abnormal. Ultrastructural investigations showed the presence of pleiomorphic enlarged lysosomes appearing as clear vacuoles containing a finely fibrillo-granular material characteristic of the visceral lysosomal storage of gangliosidoses.     

Dose dependent actions of LCL521 on acid ceramidase and key sphingolipid metabolites

The function of acid ceramidase (ACDase), whose congenital deficiency leads to Farber disease, has been recognized to be vital to tumor cell biology, and inhibition of its activity may be beneficial in cancer therapy. Therefore, manipulation of the activity of this enzyme may have significant effect, especially on cancer cells. LCL521, Di-DMG-B13, is a lysosomotropic inhibitor of ACDase. Here we define complexities in the actions of LCL521 on ACDase. Systematic studies in MCF7 cells showed dose and time divergent action of LCL521 on ACDase protein expression and sphingolipid levels. Low dose of LCL521 (1?µM) effectively inhibited ACDase in cells, but the effects were transient. A higher dose of LCL521 (10?µM) caused a profound decrease of sphingosine and increase of ceramide, but additionally affected the processing and regeneration of the ACDase protein, with biphasic and reversible effects on the expression of ACDase, which paralleled the long term changes of cellular sphingosine and ceramide. Finally, the higher concentrations of LCL521 also inhibited Dihydroceramide desaturase (DES-1). In summary, LCL521 exhibits significant effects on ACDase in a dose and time dependent manner, but dose range and treatment time need to be paid attention to specify its future exploration on ACDase targeted cancer treatment.     

A fluorescence-based,high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann-Pick disease

Acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, EC 3.1.4.12) is the lysosomal enzyme that hydrolyzes sphingomyelin (SPM) to phosphorylcholine and ceramide. An inherited deficiency of ASM activity results in Types A and B Niemann-Pick disease (NPD). In this study we report a new assay method to detect ASM activity and diagnose NPD using the fluorescent substrate BODIPY C12-SPM and reverse-phase high-performance liquid chromatography (HPLC). The reaction product, BODIPY C12-ceramide (B12Cer), could be clearly and efficiently separated from the substrate within 4 min using a reverse-phase column (Aquasil C18, Keystone Scientific). Femtomole quantities of B12Cer could be detected in as little as 1.0 micro l of human plasma, providing a sensitive measure of ASM activity. The mean ASM activity in human plasma from NPD patients (36 pmol/ml/h) was only 2.7% of that in normal plasma (1334 pmol/ml/h), confirming the specificity and diagnostic value of this new assay method. Importantly, the mean ASM activity in human plasma from NPD carriers (258.3 pmol/ml/h) also was significantly reduced (19.5% of normal). The ranges of ASM plasma activities in NPD patients (N=19), NPD carriers (N=11), and normal subjects (N=15) were 2.5-97.3, 108-551, and 1030-2124 pmol/ml/h, respectively. Based on these results, we suggest that this fluorescence-based HPLC assay method is a reliable, rapid, and highly sensitive technique to determine ASM activity and that plasma is a very reliable and simple source for the accurate diagnosis of NPD patients and carriers based on ASM activity.     

Novel analogs of D-e-MAPP and B13. Part 1: synthesis and evaluation as potential anticancer agents

A series of novel isosteric analogs of the ceramidase inhibitors, (1S,2R)-N-myristoylamino-phenylpropanol-1 (d-e-MAPP) and (1R,2R)-N-myristoylamino-4'-nitro-phenylpropandiol-1,3 (B13), with modified targeting and physicochemical properties were designed, synthesized, and evaluated as potential anticancer agents. When MCF7 cells were treated with the analogs, results indicated that the new analogs were of equal or greater potency compared to the parent compounds. Their activity was predominantly defined by the nature of the modification of the N-acyl hydrophobic interfaces: N-acyl analogs (class A), urea analogs (class B), N-alkyl analogs (class C, lysosomotropic agents), and omega-cationic-N-acyl analogs (class D, mitochondriotropic agents). The most potent compounds belonged to either class D, the aromatic ceramidoids, or to class C, the aromatic N-alkylaminoalcohols. Representative analogs selected from this study were also evaluated by the National Cancer Institute In Vitro Anticancer Drug Discovery Screen. Again, results showed a similar class-dependent activity. In general, the active analogs were non-selectively broad spectrum and had promising activity against all cancer cell lines. However, some active analogs of the d-e-MAPP family were selective against different types of cancer. Compounds LCL85, LCL120, LCL385, LCL284, and LCL204 were identified to be promising lead compounds for therapeutic development.     

Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 12–24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was $\text{s}\text{1}\text{?}\text{0\%}$ of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 10–15}\text{h}$) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) of > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transfort in Niemann-Pick type II disease, and may also suitable for diagnosis and carrier detection.     

8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines

Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.     

Identification and expression of five mutations in the human acid sphingomyelinase gene causing types A and B Niemann-Pick disease. Molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms.

The deficient activity of the human lysosomal hydrolase, acid sphingomyelinase (ASM, EC 3.1.4.12), results in the neuronopathic (Type A) and non-neuronopathic (Type B) forms of Niemann-Pick disease (NPD). To investigate the genetic basis of the phenotypic heterogeneity in NPD, the molecular lesions in the ASM gene were determined from three unrelated NPD patients and evaluated by transient expression in COS-1 cells. A Type A NPD patient of Asian Indian ancestry (proband 1) was homoallelic for a T to A transversion in exon 2 of the ASM gene which predicted a premature stop at codon 261 of the ASM polypeptide (designated L261X). In contrast, an unrelated Type A patient of European ancestry (proband 2) was heteroallelic for a two-base (TT) deletion in exon 2 which caused a frame-shift mutation at ASM codon 178 (designated fsL178), leading to a premature stop at codon 190, and a G to A transition in exon 3 which caused a methionine to isoleucine substitution at codon 382 (designated M382I). Transient expression of the fsL178, L261X, and M382I mutations in COS-1 cells demonstrated that these lesions did not produce catalytically active ASM, consistent with the severe neuronopathic Type A NPD phenotype. In contrast, an unrelated Type B patient of European descent (proband 3) was heteroallelic for two missense mutations, a G to A transition in exon 2 which predicted a glycine to arginine substitution at ASM codon 242 (designated G242R), and an A to G transition in exon 3 which resulted in an asparagine to serine substitution at codon 383 (designated N383S). Interestingly, the G242R allele produced ASM activity in COS-1 cells at levels about 40% of that expressed by the normal allele, thereby explaining the mild Type B phenotype of proband 3 and the high residual activity (i.e. approximately 15% of normal) in cultured lymphoblasts. In contrast, the N383S allele did not produce catalytically active enzyme. None of these five ASM mutations was detected in over 60 other unrelated NPD patients analyzed, nor were these mutations found in over 100 normal ASM alleles. Thus, small deletions or nonsense mutations which trunctated the ASM polypeptide, or missense mutations that rendered the enzyme noncatalytic, resulted in Type A NPD disease, whereas a missense mutation that produced a defective enzyme with residual catalytic activity caused the milder nonneuronopathic Type B phenotype. These findings have facilitated genotype/phenotype correlations for this lysosomal storage disease and provided insights into the functional organization of the ASM polypeptide.     

Synthesis and bioevaluation of ω-N-amino analogs of B13

Novel ω-N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid ceramidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and ACDase proteolytic degradation properties of LCL204 [Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.; Liu, X.; Norris, J. S.; Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015].Representative analog LCL464, (1R,2R)-2-N-(12′-N,N-dimethylaminododecanoyl amino)-1-(4″-nitrophenyl)-1,3-propandiol, inhibited ACDase activity in vitro, with a similar potency as B13 but higher than LCL204. LCL464 caused an early inhibition of this enzyme at a cellular level corresponding to decrease of sphingosine and specific increase of C₁₄- and C₁₆-ceramide. LCL464 did not induce lysosomal destabilization nor degradation of ACDase, showed increased cell death demonstrating inherent anticancer activity in a wide range of different cancer cell lines, and induction of apoptosis via executioner caspases activation. LCL464 represents a novel structural lead as chemotherapeutic agent acting via the inhibition of ACDase.     

beta-Glucosidase isoenzymes in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and patients with type 1 Gaucher disease

Lymphoid cell lines (LCL) from 3 adult patients with non-neuropathic Gaucher disease were established by Epstein-Barr virus (EBV) transformation and were investigated from the view of enzymology. Glucosylceramide-beta-glucosidase (GlcCer-beta-glucosidase) was present in soluble and particulate fraction of LCL from normal subjects and was deficient in type 1 Gaucher LCL; the deficiency of all molecular forms, shown by electrofocusing, indicates that they are coded by the same gene. The existence of two non-specific beta-glucosidases, one soluble (minor), the other membrane-bound (major), was demonstrated in leucocytes and LCL from normals; in Gaucher LCL, these were also present in a normal range. Characteristic properties of the non-specific membrane-bound beta-glucosidase were defined: lability at acidic pH and strong inhibitory effect by detergents. These properties allowed to discriminate it from the lysosomal GlcCer-beta-glucosidase and to define optimal assay conditions for determination of residual GlcCer-beta-glucosidase activity in Gaucher disease, using artificial substrate, without interference of non-specific membrane-bound beta-glucosidase. These results demonstrate that EBV-transformed LCL represent an accurate model system for enzymatic studies of Gaucher disease.     

Protein metabolism in the tumour-bearing mouse. Rates of protein synthesis in host tissues and in an Ehrlich ascites tumour at different stages in tumour growth.

The activity of acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) was measured in fibroblast homogenates from Niemann-Pick Type C (NPC) and Type D (NPD) patients to determine whether these cells exhibit similar defects in the regulation of cholesterol esterification. ACAT activity in normal cells cultured in the absence of serum lipoproteins responded rapidly (within 6 h) to the addition of serum and reached peak levels at 12-24 h, whereas little stimulation of activity in NPC cells was observed. In contrast, ACAT activity in NPD fibroblasts (cell lines from four different patients) began to increase between 6 and 12 h after serum addition, reaching levels up to 50% of normal values at 24 h. ACAT activity in NPC and NPD cell extracts could not be stimulated by preincubation with normal cell homogenates, nor was complementation between NPC and NPD homogenates observed. Addition of 25-hydroxycholesterol to fibroblasts cultured in delipidated serum increased ACAT activity for all three cell types, although stimulation in NPD cells was less than that observed in NPC cells. ACAT activity of deoxycholate-solubilized homogenates reconstituted into phosphatidylcholine vesicles was independent of the presence of serum lipoproteins during culture and dependent on cholesterol present in the vesicles for all cell types. However, ACAT activities of mutant fibroblasts in vesicles plus cholesterol were significantly (about 40%) lower than control levels. These results suggest that the metabolic lesions in NPC and NPD cells are biochemically distinct and that both may involve factors in addition to the availability of cholesterol substrate for the ACAT enzyme.     

A new approach for analysis of chemiluminescent kinetics of activated phagocytes in blood

Luminol chemiluminescence (LCL) is a simple, sensitive and time-saving tool to elucidate the oxidative activity of polymorphonuclear leukocytes (PMNL). In this study, a new approach for analysing the LCL kinetics, recorded from stimulated PMNL, and for a more accurate elucidation of their functional state, is proposed. This approach is based on the proposal that the LCL kinetics of stimulated PMNL is a result of time-probabilistic nature of the processes, leading to light emission. On this basis the LCL response was described by the parameters of a Poisson-type distribution. Relationships between these parameters and some factors influencing LCL kinetics (number of PMNL and erythrocytes, temperature, stirring) were investigated. Their development in time was different. It was suggested, that the two phases of the model LCL response were connected with extracellular and intracellular LCL. The terms and the advantages of this approach for analysis of phagocyte oxidative capacity are discussed.     

Compound heterozygosity at the sphingomyelin phosphodiesterase-1 (SMPD1) gene is associated with low HDL cholesterol

Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders caused by deficient activity of the enzyme acid sphingomyelinase (aSMase) and the resulting accumulation of sphingomyelin in tissues. In the present study, we investigated two family members who had been diagnosed with Type B NPD and who had a severe decrease in plasma high density lipoprotein cholesterol (HDL-C). The proband (a 48-year-old male) had an HDL-C of 0.30 mmol/l (12 mg/dl) and his sister had values of 0.45 mmol/l (17 mg/dl) with severe premature coronary artery disease (CAD). Hypertriglyceridemia was found in both cases. aSMase activity measured in skin fibroblasts appeared markedly depressed. The SMPD1 gene, coding for aSMase, was sequenced in affected subjects and all family members. Compound heterozygosity (DeltaR608 and R441X) was identified in both affected patients. Carriers of the DeltaR608 mutation tended to have moderately to severe decreased HDL-C levels, whereas carriers of the R441X mutation, although present only in young subjects (<20 years of age) had normal HDL-C levels. To investigate the cause of the low HDL-C level in these patients, we studied apoA-I-mediated cellular cholesterol efflux in fibroblasts. Unlike patients with Tangier disease, cholesterol efflux was found to be normal under the experimental conditions used in the present study. On the other hand, we observed a significant increase in the free cholesterol:esterified cholesterol ratio in HDL fraction from these patients and a decrease in endogenous lecithin-cholesterol acyltransferase (LCAT) activity, as determined by the fractional esterification rate. Taken together, these results suggest that (1) compound heterozygosity at the SMPD1 gene causes a severe decrease in aSMase activity and in HDL-C and increases the risk of CAD, (2) this lipoprotein abnormality is not attributable to defective cellular cholesterol efflux, (3) abnormal HDL composition might cause a decrease in LCAT activity and a lack of HDL maturation.     

Depletion of human NK cells with monoclonal antibodies allows the generation of cytotoxic T lymphocytes without NK-like cells in mixed cultures

In vitro stimulation of human mononuclear cells with x-irradiated autologous lymphoblastoid cell line (LCL) or allogeneic normal cells in mixed leukocyte cultures (MLC) was previously shown to result in the generation of OKT3+ OKT8+ cytotoxic T lymphocytes (CTL) lytic for allogeneic and autologous LCLs and also of natural killer- (NK) like cells that are OKT3- and primarily OKT8- and are lytic for HLA- NK-sensitive K562 cells. The origin of the NK-like cells was not previously known because, although the majority of fresh human NK cells react with monoclonal antibodies OKM1 and B73.1, lymphocytes bearing these markers are not detected several days after the onset of MLC, when NK-like cells are present. In this study, experiments were undertaken to determine whether NK-like cells generated after stimulation with x-irradiated pooled allogeneic normal cells (poolx) or with autologous LCL are derived from cells expressing antigens reactive with monoclonal antibodies OKM1 or B73.1, which react with fresh NK cells. Mononuclear cells, depleted of monocytes, were stained with OKM1 or B73.1 and fluorescein-labeled goat anti-mouse IgG. Lymphocytes depleted of OKM1+ or B73.1+ cells, by fluorescence-activated cell sorting, and lymphocytes that were stained but not sorted were stimulated for 7 days with either poolx or autologous LCL. The generation of NK-like activity was decreased at least 90% after depletion of cells reactive with OKM1 or B73.1, whereas the generation of CTL against autologous and allogeneic LCL was minimally affected. These findings show that NK-like cells generated in MLC are derived from cells that express the phenotype of fresh NK cells (OKM1+ or B73.1+) and that CTL can be generated in cultures in which relatively little NK-like activity is concomitantly detected, by depleting NK cells with monoclonal antibodies before stimulation.     

T cell growth factor from human splenic cell cultures: conditions for its production, and its utilization for maintenance of cytotoxic T cell lines

We prepared the T cell growth factor (TCGF) from human spleen cell cultures stimulated with phytohemagglutinin (PHA). Various cell culture conditions and agents supporting the active TCGF production of the spleen cells were examined. The highest TCGF activity was obtained in the supernatants under the conditions that 2 x 10(6)/ml spleen cells were stimulated with PHA for 48 hr. Production of TCGF from spleen cells depended markedly on their individual sources. Addition of indomethacin to the culture or irradiation of the responding spleen cells increased TCGF activity in the supernatant of the culture. Further, addition of irradiated cells of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) to spleen cell cultures stimulated with PHA greatly enhanced TCGF production. Human splenic TCGF facilitated the establishment of human cytotoxic T cell (Tc) lines specific for EBV-transformed LCL cells when those Tc line cells were stimulated periodically with irradiated autologous LCL cells but not with the other two types (K-562 or Molt-4) of cells. Allogeneic LCL stimulators allowed the Tc line cells to proliferate. However, Tc line cells cocultured once with allogeneic LCL stimulators no longer exhibited EBV-specificity in their cytotoxicities.     

Linkage of Niemann-Pick disease type D to the same region of human chromosome 18 as Niemann-Pick disease type C.

Niemann-Pick type II disease is a severe disorder characterized by accumulation of tissue cholesterol and sphingomyelin and by progressive degeneration of the nervous system. This disease has two clinically similar subtypes, type C (NPC) and type D (NPD). NPC is clinically variable and has been identified in many ethnic groups. NPD, on the other hand, has been reported only in descendants of an Acadian couple who lived in Nova Scotia in the early 18th century and has a more homogeneous expression resembling that of less severely affected NPC patients. Despite biochemical differences, it has not been established whether NPC and NPD are allelic variants of the same disease. We report here that NPD is tightly linked (recombination fraction .00; maximum LOD score 4.50) to a microsatellite marker, D18S480, from the centromeric region of chromosome 18q. Carstea et al. have reported that the NPC gene maps to this same site; therefore we suggest that NPC and NPD likely result from mutations in the same gene.     

An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease

Sphingomyelin is an important lipid component of cell membranes and lipoproteins which can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase, and the resultant accumulation of sphingomyelin in cells and tissues. In this paper we report a new, enzyme-based method to quantify the levels of sphingomyelin in tissues and plasma of normal individuals and NPD patients. The method utilizes sphingomyelinase from Bacillus cereus to completely hydrolyze the sphingomyelin into ceramide. Quantification of the sphingomyelin-derived ceramide is accomplished using Escherichia coli diacylglycerol (DAG) kinase and [gamma-(32)P]ATP. The resulting [(32)P]ceramide is quantified using a phosphor-imager system following TLC separation. This procedure allowed quantification of sphingomyelin over a broad range from 10 pmol to 1 nmol. To validate this assay we quantified sphingomyelin in plasma and tissues obtained from normal and NPD mice and humans. The sphingomyelin content in adult homozygous (-/-) or heterozygous (+/-) NPD mouse plasma was significantly elevated compared to that of normal mice (up to twofold). Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 40 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several type B NPD patients also were significantly elevated compared to normal individuals of the same age. Based on these results we propose that this new, enzyme-based procedure can provide sensitive and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could be a useful tool for the diagnosis of NPD and the evaluation of NPD treatment protocols, as well as for the study of ceramide-mediated apoptosis since the method provides the simultaneous determination of sphingomyelin and ceramide in the same lipid extract.     

Docosahexaenoic acid-derived neuroprotectin D1 induces neuronal survival via secretase- and PPARγ-mediated mechanisms in Alzheimer's disease models

Neuroprotectin D1 (NPD1) is a stereoselective mediator derived from the omega-3 essential fatty acid docosahexaenoic acid (DHA) with potent inflammatory resolving and neuroprotective bioactivity. NPD1 reduces Aβ42 peptide release from aging human brain cells and is severely depleted in Alzheimer's disease (AD) brain. Here we further characterize the mechanism of NPD1's neurogenic actions using 3xTg-AD mouse models and human neuronal-glial (HNG) cells in primary culture, either challenged with Aβ42 oligomeric peptide, or transfected with beta amyloid precursor protein (βAPP)(sw) (Swedish double mutation APP695(sw), K595N-M596L). We also show that NPD1 downregulates Aβ42-triggered expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and of B-94 (a TNF-α-inducible pro-inflammatory element) and apoptosis in HNG cells. Moreover, NPD1 suppresses Aβ42 peptide shedding by down-regulating β-secretase-1 (BACE1) while activating the α-secretase ADAM10 and up-regulating sAPPα, thus shifting the cleavage of βAPP holoenzyme from an amyloidogenic into the non-amyloidogenic pathway. Use of the thiazolidinedione peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the irreversible PPARγ antagonist GW9662, and overexpressing PPARγ suggests that the NPD1-mediated down-regulation of BACE1 and Aβ42 peptide release is PPARγ-dependent. In conclusion, NPD1 bioactivity potently down regulates inflammatory signaling, amyloidogenic APP cleavage and apoptosis, underscoring the potential of this lipid mediator to rescue human brain cells in early stages of neurodegenerations.