Lipid dynamics in neurons.

Compared with other organs, the brain is highly enriched in cholesterol. Essentially all cholesterol in the brain is synthesized within the brain; the blood-brain barrier prevents the import of plasma lipoproteins into the brain. Consequently, the brain operates an independent lipoprotein transport system in which glial cells produce ApoE (apolipoprotein E)-containing lipoproteins that are thought to deliver cholesterol to neurons for axonal growth and repair. We have shown that ApoE-containing lipoproteins generated by glial cells stimulate axon extension. ApoE associated with lipoprotein particles, and a receptor of the low-density lipoprotein receptor family, are required for stimulation of axon growth. NPC (Niemann-Pick type C) disease is a severe neurological disorder caused by mutations in the NPC1 or NPC2 gene. A hallmark of this disease is impaired transport of cholesterol out of late endosomes/lysosomes and the accumulation of cholesterol in these organelles. Although cholesterol accumulates in cell bodies of neurons from NPC1-deficient mice, the cholesterol content of axons is reduced. The presence of NPC1 in endosomal structures in nerve terminals, and the finding of aberrant synaptic vesicles, suggest that defects in synaptic vesicle recycling contribute to neurological abnormalities characteristic of NPC disease. We have also shown that ApoE-containing lipoproteins produced by glial cells from NCP1-deficient mice are of normal composition and stimulate axon extension.

Adenovirus expressing an NPC1-GFP fusion gene corrects neuronal and nonneuronal defects associated with Niemann pick type C disease.

Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by abnormal cholesterol metabolism and accumulation in lysosomal and endosomal compartments. Although peripheral organs are affected, the progressive neurodegeneration in the brain is typically most deleterious, leading to dystonia, ataxia, seizures, and premature death. Although the two genes underlying this disorder in humans and mouse models of the disease have been identified (NPC1 in 95% and NPC2/HE1 in 5% of human cases), their cellular roles have not Been fully defined, and there is currently no effective treatment for this disorder. To help address these issues, we constructed a recombinant adenovirus, Ad(NPC1-GFP), which contains a cDNA encoding a mouse NPC1 protein with a green fluorescent protein (GFP) fused to its C-terminus. Fluorescence microscopy and cholesterol trafficking assays demonstrate that the GFP-tagged NPC1 protein is functional and detectable in cells from different species (hamster, mouse, human) and of different types (ovary-derived cells, fibroblasts, astrocytes, neurons from peripheral and central nervous systems) in vitro. Combined with results from time-lapse microscopy and in vivo brain injections, our findings suggest that this adenovirus offers advantages for expressing NPC1 and analyzing its cellular localization, movement, functional properties, and beneficial effects in vitro and in vivo.

The human breast carcinoma cell line HBL-100 acquires exogenous cholesterol from high-density lipoprotein via CLA-1 (CD-36 and LIMPII analogous 1)-mediated selective cholesteryl ester uptake.

Aberrant cell proliferation is one of the hallmarks of carcinogenesis, and cholesterol is thought to play an important role during cell proliferation and cancer progression. In the present study we examined the pathways that could contribute to enhanced proliferation rates of HBL-100 cells in the presence of apolipoprotein E-depleted high-density lipoprotein subclass 3 (HDL(3)). When HBL-100 cells were cultivated in the presence of HDL(3) (up to 200 microg/ml HDL(3) protein), the growth rates and cellular cholesterol content were directly related to the concentrations of HDL(3) in the culture medium. In principle, two pathways can contribute to cholesterol/cholesteryl ester (CE) uptake from HDL(3), (i) holoparticle- and (ii) scavenger-receptor BI (SR-BI)-mediated selective uptake of HDL(3)-associated CEs. Northern- and Western-blot analyses revealed the expression of CLA-1 (CD-36 and LIMPII analogous 1), the human homologue of the rodent HDL receptor SR-BI. In line with CLA-1 expression, selective uptake of HDL(3)-CEs exceeded HDL(3)-holoparticle uptake between 12- and 58-fold. Competition experiments demonstrated that CLA-1 ligands (oxidized HDL, oxidized and acetylated low-density lipoprotein and phosphatidylserine) inhibited selective HDL(3)-CE uptake. In line with the ligand-binding specificity of CLA-1, phosphatidylcholine did not compete for selective HDL(3)-CE uptake. Selective uptake was regulated by the availability of exogenous cholesterol and PMA, but not by adrenocorticotropic hormone. HPLC analysis revealed that a substantial part of HDL(3)-CE, which was taken up selectively, was subjected to intracellular hydrolysis. A potential candidate facilitating extralysosomal hydrolysis of HDL(3)-CE is hormone-sensitive lipase, an enzyme which was identified in HBL-100 cells by Western blots. Our findings demonstrate that HBL-100 cells are able to acquire HDL-CEs via selective uptake. Subsequent partial hydrolysis by hormone-sensitive lipase could provide 'free' cholesterol that is available for the synthesis of cellular membranes during proliferation of cancer cells.

Macrophage-enhanced formation of cholesteryl ester-core aldehydes during oxidation of low density lipoprotein.

Oxidation of low density lipoproteins (LDL) results in changes to the lipoprotein particle that are potentially pro-atherogenic. To investigate mechanisms contributing to the formation of cholesteryl ester (CE)-core aldehydes (9-oxononanoyl- and 5-oxovaleroyl-cholesterol; 9-ONC and 5-OVC, respectively) LDL was incubated in the presence of mouse macrophages (J774 cells) under different culture conditions. Here we demonstrate that the formation of core aldehydes occurs only in transition metal-containing HAM's F10 medium but not in Dulbecco's modified Eagle's medium (DMEM), independent of supplementation with iron and copper at concentrations up to ten times higher than present in HAM's F10. The antioxidative properties of DMEM could be ascribed to the higher amino acid and vitamin content as compared to HAM's F10 medium. Supplementation with these components efficiently inhibited LDL oxidation in HAM's F10. Stimulation of J774 cells with phorbol ester (PMA) resulted in significantly enhanced 9-ONC and 5-OVC formation rates that were accompanied by increased consumption of LDL cholesteryl linoleate (Ch18:2) and cholesteryl arachidonate (Ch20:4) in the cellular supernatant. In PMA (10 ng/ml) activated cells, approximately 5% of Ch18:2 contained in LDL was converted to 9-ONC and 4% of Ch20:4 was converted to 5-OVC. With respect to core aldehyde formation, lipopolysaccharide (LPS, 10 microg/ml) was a less effective stimulant as compared to PMA. Part of the core aldehydes accumulated within the cells. Our study demonstrates that i) J774 macrophages are able to promote/accelerate core aldehyde formation in HAM's F10 medium, and ii) that core aldehyde formation rates can be increased by stimulation of the cells with PMA, and, although to a lesser extent, with LPS. Finally we could show that iii) a small amount of the core aldehydes is internalized by J774 macrophages.

Synthesis of 9-oxononanoyl cholesterol by ozonization.

A new route for the preparation of 9-oxononanoyl cholesterol (5) and its stable dimethylacetal (4) is described. The core aldehyde 5 is one of the major products formed during lipid peroxidation. The synthesis starts with the ozonization of oleic acid in methanol and further reduction with dimethyl sulfide to yield 9,9-dimethoxy nonanoic acid (2a). The condensation of 2a with cholesterol is achieved with N,N'-dicyclohexylcarbodiimide in dichloromethane to give 4. Further hydrolysis of 4 with the help of an acidic ion exchange resin yields 9-oxononanoyl cholesterol.

Femtomole analysis of 9-oxononanoyl cholesterol by high performance liquid chromatography.

9-Oxononanoyl cholesterol, a cholesterol core-aldehyde formed during lipoprotein oxidation, was recently identified in advanced human atherosclerotic lesions. Here we present a rapid and sensitive HPLC method for 9-oxononanoyl cholesterol analysis. 9-Oxononanoyl cholesterol was converted to the corresponding fluorescent decahydroacridine derivative by reaction with 1,3-cyclohexanedione. The derivatives formed were purified by solid-phase extraction on C-18 columns, separated by reversed phase HPLC with isocratic elution, and detected by their fluorescence. Decahydroacridine derivatives of 9-oxononanoyl cholesterol were stable for at least 160 h. The limit of quantitation of the method presented is at the low (approximately 50) femtomole level, with an absolute limit of detection (signal: noise = 6) of 15 fmol. Intra-assay variation was < or = 5%, while inter-assay variations were between 5 and 15%, depending on the concentration of the analyte. Standard curves were linear over nearly three orders of magnitude (50 fmol-12.5 pmol). 9-Oxononanoyl cholesterol proved to be the major cholesterol core-aldehyde formed during t-BuOOH/FeSO4 oxidation of cholesteryl linoleate and Cu2+-induced LDL oxidation, findings confirmed by atmospheric pressure chemical ionization-mass spectrometry. Analysis of lipid extracts obtained from advanced human atherosclerotic lesions revealed the presence of 9-oxononanoyl cholesterol in all tissue samples analyzed (28+/-14 micromol/mol cholesterol, n = 9) despite the presence of alpha-tocopherol (4+/-1.2 mmol/mol cholesterol, n = 9).

Mechanisms of lipid peroxidation in human blood plasma: a kinetic approach.

There is strong evidence that the oxidation of plasma lipoproteins plays an important role in atherogenesis. The exact mechanisms by which lipoprotein oxidation occurs in the presence of other plasma constituents, however, remains unclear. To investigate the role of different antioxidants for this process, we studied the oxidation of human plasma supplemented in vitro with physiological amounts of major plasma antioxidants alpha-tocopherol, ubiquinol-10 ascorbate, urate, bilirubin and albumin. The plasma was diluted 2-fold and oxidized by 3.75 mM Cu(II). The concentrations of the antioxidants, fatty acids, linoleic acid hydroperoxides and oxycholesterols in oxidizing plasma were measured. The oxidation was characterized by three consecutive phases similar to the known lag, propagation, and decomposition phases of low density lipoprotein oxidation. The rate of the initiation of oxidation as calculated from antioxidant consumption rates was raised by supplementation with alpha-tocopherol or ascorbate. The oxidation rate in the lag phase was lowered by supplementation with any of the antioxidants, whereas in the propagation phase the oxidation rate was slightly higher in supplemented than in unsupplemented plasma. The kinetic chain length in the lag phase was less than one in supplemented plasma and about one in unsupplemented plasma. The chain length in the propagation phase was between three and six for all plasma samples. A higher rate of urate consumption and a reduced rate of alpha-tocopherol consumption were found in plasma supplemented with ascorbate in comparison with unsupplemented plasma. These data suggest that: (i) the reduction of Cu(II) by alpha-tocopherol and ascorbate is a major initiating event in Cu(II)-catalyzed oxidation of human plasma; (ii) the following lag phase is caused by radical-scavenging effects of all antioxidants with alpha-tocopherol as a major lipophilic and urate as a major hydrophilic scavenger; (iii) interactions between antioxidants, such as regeneration of ascorbate by urate and of alpha-tocopherol by ascorbate, take place during the lag phase; (iv) in the absence of added antioxidants the oxidation in the lag phase can occur via a chain reaction; and (v) in the propagation phase the oxidation is not inhibited by antioxidants and occurs autocatalytically.

Whole plasma oxidation assay as a measure of lipoprotein oxidizability.

Lipoprotein oxidation induced in vitro in whole plasma is expected to be a more relevant model of the lipoprotein oxidation in the arterial wall than the in vitro oxidation of single isolated lipoproteins, e.g., low density lipoprotein (LDL). However, it is unclear, whether the oxidizability of whole plasma may serve as an adequate measure of the oxidizability of plasma lipoproteins. We measured the oxidizability of whole plasma diluted 150-fold as an absorbance increase at 234 nm known to reflect the level of conjugated dienes in the samples. Plasma oxidation was induced by Cu(II), 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), lipoxygenase or myeloperoxidase+H2O2. Oxidizability of human plasma measured in the presence of Cu(II) was found to correlate with the oxidizability of LDL measured in the common Cu(II)-based LDL oxidation assay. The plasma oxidizability also correlated positively with plasma oxidizable fatty acid and negatively with plasma antioxidant content. Supplementation of human plasma with different antioxidants (albumin, urate, ascorbate, bilirubin, alpha-tocopherol and ubiquinol-10) in vitro decreased its oxidizability. Supplementation of Watanabe heritable hyperlipidaemic rabbits with different antioxidants (vitamin E, ubiquinone-10, probucol, carvedilol) in vivo lowered the oxidizability of rabbit plasma in comparison with rabbits fed standard diet. When plasma from hyperlipidaemic patients with or without coronary heart disease and from age-matched healthy controls was studied, the plasma oxidizability was found to be highest in the patients with coronary heart disease and lowest in the controls. Taken together, these data indicate that the plasma oxidation assay (i) provides information similar to that obtained using the common LDL oxidation assay, (ii) upgrades the latter, taking into account the effect of hydrophilic antioxidants on lipoprotein oxidation and characterizing the oxidizability of all plasma lipoproteins, and (iii) offers important practical advantages, such as fast and simple sample processing, low amount of plasma required and avoidance of artefactual oxidation during lipoprotein isolation. We propose the measurement of plasma oxidizability at 234 nm as an adequate practical index of the oxidizability of plasma lipoproteins.

Antioxidant and prooxidant activity of alpha-tocopherol in human plasma and low density lipoprotein.

Alpha-Tocopherol is a classical lipophilic antioxidant well known as a scavenger of free radicals in a hydrophobic milieu. However, it can develop both anti- and prooxidant activity in isolated low density lipoprotein (LDL). It is unknown how these activities are balanced in vivo in human plasma. We studied oxidation of plasma and LDL isolated from healthy donors or from a patient with familial isolated vitamin E deficiency and supplemented with alpha-tocopherol in vivo or in vitro. We found that alpha-tocopherol supplementation decreased plasma and LDL oxidizability under strong oxidative conditions when oxidation was initiated by high amounts of Cu2+ or 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH). The effect was independent of the presence of ascorbate in the samples. Under conditions of mild oxidation by low amounts of Cu2+ or AAPH, alpha-tocopherol supplementation decreased plasma oxidizability only in the presence of physiological amounts of ascorbate. A prooxidant effect of alpha-tocopherol was found under mild oxidative conditions in highly diluted (150-fold) plasma and in isolated LDL. These results indicate that the level of oxidative stress and concentration of co-antioxidants, such as ascorbate, capable of regenerating alpha-tocopherol in the oxidizing lipoprotein particle, appear to represent major factors determining alpha-tocopherol activity towards oxidation both in human plasma and LDL. In vivo, in the presence of high concentrations of co-antioxidants and under mild oxidative conditions, alpha-tocopherol should normally behave as an antioxidant. This antioxidant activity is also expected to prevail under strong oxidative conditions independently of the presence of co-antioxidants but it may evolve into prooxidant, when the co-antioxidants are exhausted under conditions of mild oxidation. It remains to be shown whether such a transformation is physiologically relevant and can occur in vivo.