Alterations of cholesterol synthesis precursors (7-dehydrocholesterol, 7-dehydrodesmosterol, desmosterol) in dysmyelinating neurological mutant mouse (quaking, shiverer and trembler) in the PNS and the CNS

In brain, levels of cholesterol, desmosterol and 7-dehydrodesmosterol are reduced in shiverer and quaking, but not in trembler 60-day-old dysmyelinating mutant mice. Very interestingly, 7-dehydrocholesterol is not altered in any mutant. The amount of cholesterol is similar in the different normal control mouse strains and in rat. In contrast, levels of precursors are not the same. In sciatic nerve, cholesterol is slightly reduced in shiverer, reduced 2-fold in quaking, and dramatically reduced in trembler (10-fold). 7-Dehydrocholesterol is affected in all mutants.     

Alteration of α-Tocopherol Content in the Developing and Aging Peripheral Nervous System: Persistence of High Correlations with Total and Specific (n-6) Polyunsaturated Fatty Acids

In contrast to brain, the sciatic nerve concentration of vitamin E in rats increased rapidly during the postnatal period (approximately fivefold between days 1 and 8), then decreased dramatically (about twofold between days 8 and 30), and further decreased slowly between days 30 and 60 and remained constant up to 2 years. Although the sciatic nerve concentration of vitamin E decreased by 58% between days 8 and 30, the concentration of vitamin E in serum presented a marked decrease (approximately 75%). The vitamin E concentrations varied in a similar pattern in whole sciatic nerve and in endoneurium and showed a very close correlation (r = 0.94). The age-related changes in fatty acid concentration of the endoneurial fraction of the sciatic nerve were characterized by a large increase in content of saturated and monounsaturated fatty acids up to 6 months (twofold for saturated and fourfold for monounsaturated fatty acids). Then, up to 24 months, the amount of these fatty acids decreased very slowly. The content of (n-6) polyunsaturated fatty acids (PUFAs) decreased rapidly up to 1 year and slowly afterward. In contrast, during development the amount of (n-3) PUFA was relatively stable and decreased during aging. A highly significant correlation between vitamin E and (n-6) PUFA [18:2(n-6), 20:4(n-6), and total (n-6)] was observed but not between (n-3) PUFA and vitamin E. It is suggested that there may be a relationship between vitamin E and (n-6) PUFA in the PNS membranes during development and aging.     

Oxidation of 7-dehydrocholesterol and desmosterol by human cytochrome P450 46A1

Cytochrome P450 (P450 or CYP) 46A1 is expressed in brain and has been characterized by its ability to oxidize cholesterol to 24S-hydroxycholesterol. In addition, the same enzyme is known to further oxidize 24S-hydroxycholesterol to the 24,25- and 24,27-dihydroxy products, as well as to catalyze side-chain oxidations of 7α-hydroxycholesterol and cholestanol. As precursors in the biosynthesis of cholesterol, 7-dehydrocholesterol has not been found to be a substrate of P450 46A1 and desmosterol has not been previously tested. However, 24-hydroxy-7-dehydrocholesterol was recently identified in brain tissues, which prompted us to reexamine this enzyme and its potential substrates. Here we report that P450 46A1 oxidizes 7-dehydrocholesterol to 24-hydroxy-7-dehydrocholesterol and 25-hydroxy-7-dehydrocholesterol, as confirmed by LC-MS and GC-MS. Overall, the catalytic rates of formation increased in the order of 24-hydroxy-7-dehydrocholesterol < 24-hydroxycholesterol < 25-hydroxy-7-dehydrocholesterol from their respective precursors, with a ratio of 1:2.5:5. In the case of desmosterol, epoxidation to 24S,25-epoxycholesterol and 27-hydroxylation was observed, at roughly equal rates. The formation of these oxysterols in the brain may be of relevance in Smith-Lemli-Opitz syndrome, desmosterolosis, and other relevant diseases, as well as in signal transduction by lipids.     

Altered Expression of Inducible Nitric Oxide Synthase After Sciatic Nerve Injury in Rat

Nitric oxide is known to contribute to neuronal damage as well as to peripheral neuronal regeneration following injury. Sciatic nerve injury is a common and serious complication of intramuscular injections. In order to ascertain the role of inducible nitric oxide synthase (iNOS) in the injured sciatic nerve, we studied the expression of this enzyme by RT-PCR and immunohistochemistry, in a rat model of sciatic nerve injury. In sham-operated control rats iNOS expression was undetectable by immunohistochemistry and its mRNA level was also very low. In contrast, in the experimental group that was subjected to sciatic nerve injury, both mRNA and protein of iNOS were found to be significantly elevated. The protein level of iNOS, as revealed by positive immunostaining, peaked at 7 days post-surgery followed by a decrease. Similarly, the iNOS mRNA levels remained elevated at 1, 3, 7 days but declined to very low level by day 21, after surgery. This study indicates that the increased expression of iNOS after sciatic nerve injury in rats may contribute to nerve regeneration. Thus our results suggest that excessive expression of iNOS after nerve injury is not conducive to nerve regeneration.     

Tellurium-Induced Alterations in 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene Expression and Enzyme Activity: Differential Effects in Sciatic Nerve and Liver Suggest Tissue-Specific Regulation of Cholesterol Synthesis

The demyelination of peripheral nerves that results from exposure of developing rats to tellurium is due to inhibition of squalene epoxidase, a step in cholesterol biosynthesis. In sciatic nerve, cholesterol synthesis is greatly depressed, whereas in liver, some compensatory mechanism maintains normal levels of cholesterol synthesis. This tissue specificity was further explored by examining, in various tissues, gene expression and enzyme activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Exposure to tellurium resulted in pronounced increases in both message levels and enzyme activity in liver, the expected result consequent to up-regulation of this enzyme in response to decreasing levels of intracellular sterols. In contrast to liver, levels of mRNA and enzyme activity in sciatic nerve were both decreased during the tellurium-induced demyelinating period. The temporal pattern of changes in 3-hydroxy-3-methylglutaryl-CoA reductase message levels in sciatic nerve seen following exposure to tellurium was similar to the down-regulation seen for mRNA specific for PNS myelin proteins. Possible mechanisms for differential control of cholesterol biosynthesis in sciatic nerve and liver are discussed.     

Cholesterol biosynthesis from birth to adulthood in a mouse model for 7-dehydrosterol reductase deficiency (Smith-Lemli-Opitz syndrome)

Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis, which is catalyzed by 7-dehydrocholesterol reductase (DHCR7). The disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. Pathogenesis of SLOS is complex due to multiple roles of cholesterol and may be further complicated by unknown effects of aberrant metabolites that arise when 7-dehydrocholesterol (7-DHC), the substrate for DHCR7, accumulates. A viable mouse model for SLOS has recently been developed, and here we characterize cholesterol metabolism in this model with emphasis on changes during the first few weeks of postnatal development. Cholesterol and 7-DHC were measured in "SLOS" mice and compared with measurements in normal mice. SLOS mice had measurable levels of 7-DHC at all ages tested (up to 1 year), while 7-DHC was below the threshold for detection in normal mice. In perinatal to weaning age SLOS mice, cholesterol and 7-DHC levels changed dramatically. Changes in brain and liver were independent; in brain cholesterol increased several fold while 7-DHC remained relatively constant, but in liver cholesterol first increased then decreased again while 7-DHC first decreased then increased. In older SLOS animals the ratio of 7-DHC/cholesterol, which is an index of biochemical severity, tended to approach, but not reach, normal. While these mice provide the best available genetic animal model for the study of SLOS pathogenesis and treatment, they probably will be most useful at early ages when the metabolic effects of the mutations are most dramatic. To correlate any experimental treatment with improved sterol metabolism will require age-matched controls. Finally, determining the mechanism by which these "SLOS" mice tend to normalize may provide insight into the future development of therapy.     

Cholesterol Synthesis Is Down-Regulated During Regeneration of Peripheral Nerve

Abstract: The discovery of apolipoprotein E synthesis and secretion by injured peripheral nerve led to the hypothesis that endoneurial apolipoprotein E serves to salvage degenerating myelin cholesterol. This salvaged cholesterol could then be reutilized by Schwann cells during remyelination via uptake through low-density lipoprotein receptors. As a test of this hypothesis, we measured the rate of cholesterol synthesis in rat sciatic nerve endoneurium during development and at various times following a crush injury at 50 days of age. In control nerves [¹⁴C]acetate incorporation into cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase activity were closely linked throughout development, indicating that reductase activity in nerve, as in other tissues, is a good indicator of cholesterol's synthetic rate. In the crushed nerves cholesterol synthesis fell to nearly zero during the first week after the crush. There was a partial recovery during the second to fourth weeks, but unlike that of other lipids, cholesterol synthesis remained well below control nerve values throughout most of the 15-week post-crush period examined. Thus, cholesterol synthesis is at very low levels during the myelination of regenerating axons. These results are consistent with a receptor-mediated down-regulation of cholesterol synthesis by lipoproteins, and would be expected if Schwann cells were utilizing an external source of cholesterol as postulated above.     

A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome

We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD “ene” reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.     

Age-related variations of elements as compared among optic, radial, and sciatic nerves

To elucidate changes of peripheral nerves with aging, the authors studied age-related changes of element contents in the optic, radial, and sciatic nerves by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. The contents of phosphorus and sulfur remained constant through ages 61 to 97 yr in three nerves, the optic, radial, and sciatic nerves. It was found that there were age-related differences in calcium content among the optic, radial, and sciatic nerves: The calcium content of the optic nerve increased progressively with aging; in the radial nerve, it was hardly changed with aging; in contrast, the calcium content of the sciatic nerve decreased gradually with aging. In addition, it was found that in the radial nerve there were moderate correlations between age and zinc or sodium content, whereas significant correlations between age and the content of silicon or iron were found in the sciatic nerve. Furthermore, there was a correlation between the silicon and iron contents in the sciatic nerves.     

Cholesterol esters and hydrolytic cholesterol esterase during Wallerian degeneration

To study the involvement of cholesterol esters in myelination and demyelination, we determined the concentration of free cholesterol and cholesterol esters and the activity of hydrolytic cholesterol esterase (sterol ester hydrolase; EC 3.1.1.13) in hen sciatic nerve during Wallerian degeneration. A progressive increase in the ratio of cholesterol ester to free cholesterol was observed in the degenerating nerve at 8, 16 and 32 days after nerve section. Hydrolytic cholesterol esterase activity decreased progressively in the degenerating nerves at the same time. In addition we measured the ratio of RNA to DNA, and the activity of the NADP⁺-dependent isocitrate dehydrogenase [L₈-isocitrate: NADP oxidoreductase (decarboxylating); EC 1.1.1.42] at 8, 16 and 32 days after nerve section. The RNA to DNA ratios decreased progressively in the degenerating nerves. NADP⁺-dependent isocitrate dehydrogenase increased in activity after nerve section, reaching a peak at 16 days.     

Role of sterol type on lateral pressure profiles of lipid membranes affecting membrane protein functionality: Comparison between cholesterol, desmosterol, 7-dehydrocholesterol and ketosterol

Lateral pressure profiles have been suggested to play a significant role in many cellular membrane processes by affecting, for example, the activation of membrane proteins through changes in their conformational state. This may be the case if the lateral pressure profile is altered due to changes in molecular composition surrounding the protein. In this work, we elucidate the effect of varying sterol type on the lateral pressure profile, an issue of topical interest due to lipid rafts and their putative role for membrane protein functionality. We find that the lateral pressure profile is altered when cholesterol is replaced by either desmosterol, 7-dehydrocholesterol, or ketosterol. The observed changes in the lateral pressure profile are notable and important since desmosterol and 7-dehydrocholesterol are the immediate precursors of cholesterol along its biosynthetic pathway. The results show that the lateral pressure profile and the resulting elastic behavior of lipid membranes are sensitive to the sterol type, and support a mechanism where changes in protein conformational state are facilitated by changes in the lateral pressure profile. From a structural point of view, the results provide compelling evidence that despite seemingly minor differences, sterols are characterized by structural specificity.     

Effect of increasing amounts of dietary fish oil on brain and liver fatty composition

Increasing dietary fish oil in rat had the following effect on brain lipids: Arachidonic acid regularly decreased; eicosapentanenoic acid, normally nearly undetectable, was present; 22:5(n - 3), dramatically increased but remained below 1% of total fatty acids; cervonic acid was increased by 30% at high fish oil concentration. Saturated and monounsaturated fatty acids were not affected regardless of chain-length. In contrast, in the liver, nearly all fatty acids (saturated, monounsaturated and polyunsaturated) were affected by high dietary content of fish oil, but liver function was normal: serum vitamin A and E, glutathione peroxidase, alkaline phosphatase, transaminases were not affected. Serum total cholesterol, unesterified cholesterol and phosphatidylcholine were slightly affected. In contrast, triacylglycerols were dramatically reduced in proportion to the fish oil content of the diet.     

Effect of long-term administration of AY-9944, an inhibitor of 7-dehydrocholesterol delta 7-reductase, on serum and tissue lipids in the rat

The effect of long-term administration of AY-9944, a specific inhibitor of cholesterol biosynthesis, was examined in rats maintained on diets with low and high cholesterol and fat content. Sterol and phospholipid levels were determined in the serum, liver, adrenals, lungs, and brain after 6 and 12 months of feeding AY-9944 at several dose levels. In all the tissues examined, the cholesterol content was lowered and the cholesterol was partly replaced by 7-dehydrocholesterol biosynthesized instead of cholesterol in the presence of AY-9944. Cholesterol levels were particularly low in the serum and adrenals, while 7-dehydrocholesterol accumulated in the lungs. The fall in cholesterol and appearance of 7-dehydrocholesterol were reversible. Alterations of this type in the brain indicated that sterol metabolism is active in the adult rat brain. Addition of cholesterol to the diet reduced the effect of the inhibitor by eliminating the liver as a site of sterol synthesis.     

Inhibition of human fibroblasts sphingomyelinase by cholesterol and 7-dehydrocholesterol

An inhibition of human fibroblast sphingomyelinase by cholesterol and 7-dehydrocholesterol is shown. This effect is obtained for cholesterol and 7-dehydrocholesterol/sphingomyelin molar ratios above 0.1. Diffusion measurements performed on mixed liposomes demonstrated for cholesterol/sphingomyelin and 7-dehydrocholesterol/sphingomyelin molar ratios above 0.1 a sharp increase in diffusion intensity. The mechanism of the inhibition of sphingomyelinase by sterols is discussed in relation to the physical state of the substrate. A possible involvement of this phenomenon in sphingomyelin accumulation observed in aging or in atheroma is discussed.     

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC > cholesterol > desmosterol > lanosterol > epicholesterol > 4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol > lanosterol ~ desmosterol ~ epicholesterol or prior studies in which the ranking was cholesterol ~ epicholesterol > lanosterol ~ desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3β-OH group.     

Neutral sterols of rat epididymis. High concentrations of dehydrocholesterols in rat caput epididymidis.

Phospholipids and sterols are known to have multiple functions in reproductive tissue of mammals. High concentrations of the cholesterol precursor desmosterol have been described in testis, epididymis, and spermatozoa of various species. These findings and the recent discovery of some cholesterol precursors as meiosis-activating sterols suggest important functions of cholesterol precursors in fertility. Many sterol intermediates appear from the 19-step conversion of lanosterol, the first sterol synthesized in the cascade of cholesterol synthesis, to cholesterol. The biochemical basis of the genetically inherited Smith-Lemli-Opitz syndrome has been described as a defective conversion of 7-dehydrocholesterol to cholesterol. Since this discovery, interest has focused on this special cholesterol precursor. Here, we report high concentrations of 7- and 8-dehydrocholesterol in caput epididymidis and spermatozoa derived from caput epididymidis of Sprague-Dawley and Wistar rats, which comprised up to 30% of total sterols. In contrast to caput epididymidis, 7- and 8-dehydrocholesterol were barely detected in cauda epididymidis or testis. Desmosterol increased several times from caput to cauda epididymidis.This is the first report of the natural appearance of high concentrations of dehydrocholesterols in mammalian tissue, and it underlines the putative importance of cholesterol precursors in reproductive tissue.     

Regulation of UDP-Galactose:Ceramide Galactosyltransferase and UDP-Glucose:Ceramide Glucosyltransferase After Crush and Transection Nerve Injury

The enzyme activities of ceramide galactosyltransferase and ceramide glucosyltransferase were assayed as a function of time (0, 1, 2, 4, 7, 14, 21, 28, and 35 days) after crush injury or permanent transection of the adult rat sciatic nerve. These experimental models of neuropathy are characterized by the presence and absence of axonal regeneration and subsequent myelin assembly. Within the first 4 days after both injuries, a 50% reduction of ceramide galactosyltransferase-specific activity was observed compared to values found in the normal adult nerve. This activity remained unchanged at 7 days after injury; however, by 14 days the ceramide galactosyltransferase activity diverged in the two models. The activity increased in the crushed nerve and reached control values by 21 days, whereas a further decrease was observed in the transected nerve such that the activity was nearly immeasurable by 35 days. In contrast, the ceramide glucosyltransferase activity showed a rapid increase between 1 and 4 days, followed by a plateau that was 3.4-fold greater than that in the normal adult nerve, which persisted throughout the observation period in both the crush and transection models. [3H]Galactose precursor incorporation studies at 7, 14, 21, and 35 days after injury confirmed the previously observed shift in biosynthesis from the galactocerebrosides during myelin assembly in the crush model to the glucocerebrosides and oligohexosylceramide homologues in the absence of myelin assembly in the transection model. The transected nerves were characterized by a peak of biosynthesis of the glucocerebrosides at 14 days. Of particular interest is the biosynthesis of the glucocerebrosides and the oligohexosylceramides at 7 and 14 days after crush injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS

Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane.     

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.