Lipidomic analysis of the retina in a rat model of Smith-Lemli-Opitz syndrome: alterations in docosahexaenoic acid content of phospholipid molecular species

Smith–Lemli–Opitz syndrome (SLOS) is a complex hereditary disease caused by an enzymatic defect in the last step of cholesterol biosynthesis. Progressive retinal degeneration occurs in an AY9944-induced rat model of SLOS, with biochemical and electroretinographic hallmarks comparable with the human disease. We evaluated alterations in the non-sterol lipid components of the retina in this model, compared with age-matched controls, using lipidomic analysis. The levels of 16:0–22:6 and 18:0–22:6 phosphatidylcholine molecular species in retinas were less by > 50% and > 33%, respectively, in rats treated for either 2 or 3 months with AY9944. Relative to controls, AY9944 treatment resulted in > 60% less di-22:6 and > 15% less 18:0–22:6 phosphatidylethanolamine molecular species. The predominant phosphatidylserine (PS) molecular species in control retinas were 18:0–22:6 and di-22:6; notably, AY9944 treatment resulted in > 80% less di-22:6 PS, relative to controls. Remarkably, these changes occurred in the absence of n3 fatty acid deficiency in plasma or liver. Thus, the retinal lipidome is globally altered in the SLOS rat model, relative to control rats, with the most profound changes being less phosphatidylcholine, phosphatidylethanolamine, and PS molecular species containing docosahexaenoic acid (22:6). These findings suggest that SLOS may involve additional metabolic compromise beyond the primary enzymatic defect in the cholesterol pathway.     

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith–Lemli–Opitz syndrome

Smith–Lemli–Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ⁷-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d₇-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25 μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.     

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ(7)-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d(7)-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.     

Formation of 7-dehydrocholesterol-containing membrane rafts in vitro and in vivo, with relevance to the Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease typified by 7-dehydrocholesterol (7DHC) accumulation and depletion of cholesterol. Because cholesterol is a primary component of detergent-resistant membrane domains ("rafts"), we examined the compatibility of 7DHC with raft formation. Liposomes containing bovine brain phosphatidylcholine, sphingomyelin, cerebrosides, and either cholesterol, 7DHC, or coprostanol (the latter being incompatible with raft formation) were prepared. 7DHC was indistinguishable from cholesterol in its ability to become incorporated into membrane rafts, as judged by physical and chemical criteria, whereas coprostanol did not form rafts. The in vivo compatibility of 7DHC with raft formation was evaluated in brains of rats treated with trans-1,4-bis(2-dichlorobenzylamino-ethyl)cyclohexane dihydrochloride (AY9944), which mimics the SLOS biochemical defect. 7DHC/cholesterol ratios in rafts and whole brains from AY9944-treated rats were similar, indicating comparable efficiency of 7DHC and cholesterol incorporation into brain rafts. In contrast, dolichol (a nonsterol isoprenoid incompatible with raft formation) was greatly depleted in brain rafts relative to whole brain. Although brain raft fractions prepared from AY9944-treated and control rats yielded similar sterol-protein ratios, their gel electrophoresis profiles exhibited multiple differences, suggesting that altered raft sterol composition perturbs raft protein content. These results are discussed in the context of the SLOS phenotype, particularly with regard to the associated central nervous system defects.     

Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz Syndrome

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.     

Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency

The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had approximately 80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (G(t)) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-G(t) complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency.     

Signaling by the human serotonin1A receptor is impaired in cellular model of Smith-Lemli-Opitz Syndrome

The Smith-Lemli-Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin_1A receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin_1A receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin_1A receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin_1A receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin_1A receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.     

Effect of docosahexaenoic acid and ascorbate on peroxidation of retinal membranes of ODS rats

Mutant male osteogenic disorder Shionogi (ODS) rats, unable to synthesize ascorbic acid, were fed diets containing a high content of docosahexaenoic acid (DHA) and different amounts of ascorbic acid, to study the effect of DHA on peroxidative susceptibility of the retina and possible antioxidant action of ascorbic acid. ODS rats were fed from 7 weeks of age with diets containing high DHA (6.4% of total energy). A control group received a diet high in linoleic acid. The diets also contained varying amounts of ascorbic acid. Fatty acid compositions and phospholipid hydroperoxides in rod outer segment (ROS) membranes, and retinal ascorbic acid were analyzed. DHA in ROS membranes was significantly increased in rats fed high DHA, compared with the linoleic acid diet. Levels of phospholipid hydroperoxides in the DHA-fed rats were significantly higher than the linoleic acid-fed rats. Ascorbic acid supplementation did not suppress the phospholipid hydroperoxide levels after a high DHA diet, even when the supplement increased the content of retinal ascorbic acid. In conclusion, high DHA feeding induced a marked increase of phospholipid hydroperoxides in ROS membranes of ODS rats. Supplementation of ascorbic acid did not reverse this increase.     

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (α-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%～5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.     

Novel oxysterols observed in tissues and fluids of AY9944-treated rats: a model for Smith-Lemli-Opitz syndrome

Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ(7)-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d(7)-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.     

The effect of the hypocholesteremic drug, AY 9944 on the synthesis of bile salts in rat liver

1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis.     

Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome animal model

Low cholesterol and high 7-dehydrocholesterol (7DHC) levels are associated with a blockade of Delta7-reductase in the Smith-Lemli-Opitz syndrome (SLOS) and in the animals treated with the inhibitor AY9944. The impact of the cholesterol deficit and of the accumulation of 7DHC on the embryo were investigated in AY9944-treated pregnant rats receiving an enriched cholesterol or 7DHC diet. Sterol profiling was performed under the various nutritional conditions. AY9944 caused a severe decrease in the maternal and embryo cholesterol. The deficit in the embryo was sustained by the embryonic uptake of the inhibitor. A cholesterol-rich diet was efficient in restoring the maternal and embryonic cholesterol and phenotype but a 7DHC-rich diet did not modify the sterol status compared with dams treated with only AY9944. The offspring phenotype remained deleterious whether or not the dams received 7DHC-rich diet. Over 80% of the 7DHC was absorbed, as was cholesterol, which was not quantitatively influenced by AY9944. When cholesterol and 7DHC were simultaneously administered, a competition for intestinal absorption enhanced the lowering cholesterol effect of AY9944.Whether or not the dams received a 7DHC dietary supplement, the offspring's phenotype became normal when the diet was supplemented with cholesterol. Under conditions in which the ratio of cholesterol/7DHC is substantially varied, the normal development of embryos can be achieved as long as the cholesterol is sufficient. The phenotype is reversed in vivo by cholesterol which contrasts with the irreversible effects manifested in vitro by oxidized 7DHC by-products.     

Docosahexaenoic acid affects insulin deficiency- and insulin resistance-induced alterations in cardiac mitochondria

The effect of docosahexaenoic acid (DHA) intake on cardiac mitochondrial function was evaluated in permeabilized fibers in insulin deficiency and insulin resistance in rats. The insulin-deficient state was obtained by streptozotocin injection 2 mo before investigations. Insulin resistance was obtained by feeding a 62% fructose diet for 3 mo. DHA was incorporated in the diet to modify the fatty acid composition of cardiac membranes, including mitochondria. Insulin deficiency decreased mitochondrial creatine kinase (mi-CK) activity and mitochondrial sensitivity to ADP. DHA intake prevented these alterations. Moreover, the insulin-deficient state significantly decreased n-3 polyunsaturated fatty acids (PUFA) and slightly increased n-6 PUFA in both cardiac and mitochondrial membranes, inducing a significant increase in the n-6-to-n-3 ratio. DHA intake maintained high myocardial and mitochondrial DHA content. Insulin deficiency also decreased glutamate- and palmitoylcarnitine-supported mitochondrial respiration, but DHA intake did not prevent these effects. In contrast, insulin resistance did not affect mi-CK activity or sensitivity to ADP. However, insulin resistance influenced the myocardial fatty acid composition with decreased n-6 and n-3 PUFA contents and increased monounsaturated fatty acid content. Only slight alterations were observed in mitochondrial fatty acid composition, and they were corrected by DHA intake. Moreover, insulin resistance decreased the glutamate-supported respiration, and DHA intake did not influence this effect. In conclusion, the impairment of cardiac mitochondrial function was more pronounced in the insulin-deficient state than in insulin resistance. The modification of fatty acid composition of cardiac and mitochondrial membranes by DHA partially prevented the mitochondrial alterations induced in the two models.     

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas.

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%-5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.     

Docosahexaenoic acid but not eicosapentaenoic acid withstands dietary cholesterol-induced decreases in platelet membrane fluidity

To determine the differenetial effects of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid on platelet membrane fluidity under hypercholesterolemic conditions. DHA and EPA were orally administered (300 mg/kg body weight^.day) to hypercholesterolemic rats for 12 weeks. Membrane fluidity, evaluated by fluorescence polarization of nonpolar 1,6-diphenyl-1,3,5-hexatriene (DPH), of the platelets of high cholesterol (HC; 1%)-fed rats decreased significantly compared with that of the platelets of normocholesterolemic rats. In HC-fed rats, dietary administration of DHA, unlike that of EPA, significantly increased platelet membrane fluidity. A high cholesterol diet significantly increased platelet aggregation, compared with the platelet aggregation of normocholesterolemic rats. DHA administration significantly decreased the aggregation, whereas EPA had no effect. Levels of EPA in the platelets of the EPA-fed HC rats and those of DHA in the platelets of the DHA-fed HC rats increased by 482 and 174%, respectively, compared with those in the platelets of the HC-fed rats. The unsaturation index and the ratio of saturated to (poly)unsaturated fatty acid of the platelet membrane increased only in the DHA-fed rats. The phospholipid content in platelet membranes remained unaltered in all groups, whereas the cholesterol content decreased significantly in DHA-fed rats, resulting in a significant decrease in the cholesterol/phospholipid molar ratio only in the platelet membranes of DHA-fed rats. These results suggest that DHA is a more potent membrane-fluidizer than EPA in withstanding cholesterol-induced decreases in platelet membrane fluidity and a stronger ameliorative modulator of platelet hyperaggregation.     

Perinatal n-3 fatty acid deficiency selectively reduces myo-inositol levels in the adult rat PFC: an in vivo (1)H-MRS study

To investigate the effects of omega-3 fatty acid deficiency on phosphatidylinositol signaling in brain, myo-inositol (mI) concentrations were determined in the prefrontal cortex (PFC) of omega-3 fatty acid deficient rats by in vivo proton magnetic resonance spectroscopy ((1)H-MRS). To generate graded deficits in PFC docosahexaenoic acid (22:6n-3) (DHA) composition, perinatal and postweaning alpha-linolenic acid (18:3n-3) (ALA) deficiency models were used. Adult male rats were scanned in a 7T Bruker Biospec system and a (1)H-MRS spectrum acquired from the bilateral medial PFC. Rats were then challenged with SKF83959, a selective agonist at phosphoinositide (PI)-coupled dopamine D(1) receptors. Postmortem PFC fatty acid composition was determined by gas chromatography. Relative to controls, PFC DHA composition was significantly reduced in adult postweaning (-27%) and perinatal (-65%) ALA-deficiency groups. Basal PFC mI concentrations were significantly reduced in the perinatal deficiency group (-21%, P = 0.001), but not in the postweaning deficiency group (-1%, P = 0.86). Among all rats, DHA composition was positively correlated with mI concentrations and the mI/creatine (Cr) ratio. SKF83959 challenge significantly increased mI concentrations only in the perinatal deficiency group (+16%, P = 0.02). These data demonstrate that perinatal deficits in cortical DHA accrual significantly and selectively reduce mI concentrations and augment receptor-generated mI synthesis.     

Role of cholesterol in the regulation of growth plate chondrogenesis and longitudinal bone growth

Inborn errors of cholesterol synthesis are associated with multiple systemic abnormalities, including skeletal malformations. The regulatory role of cholesterol during embryogenesis appears to be mediated by Shh, a signaling molecule in which activity depends on molecular events involving cholesterol. Based on this evidence, we hypothesized that cholesterol, by modifying the activity of Ihh (another of the Hedgehog family proteins) in the growth plate, regulates longitudinal bone growth. To test this hypothesis, we treated rats with AY 9944, an inhibitor of the final reaction of cholesterol synthesis. After 3 weeks, AY 9944 reduced the cumulative growth, tibial growth, and the tibial growth plate height of the rats. To determine whether cholesterol deficiency affects bone growth directly at the growth plate, we then cultured fetal rat metatarsal bones in the presence of AY 9944. After 4 days, AY 9944 suppressed metatarsal growth and growth plate chondrocyte proliferation and hypertrophy. The inhibitory effect on chondrocyte hypertrophy was confirmed by the AY 9944-mediated decreased expression of collagen X. Lastly, AY 9944 decreased the expression of Ihh in the metatarsal growth plate. We conclude that reduced cholesterol synthesis in the growth plate, possibly by altering the normal activity of Ihh, results in suppressed longitudinal bone growth and growth plate chondrogenesis.     

Signaling by the human serotonin(1A) receptor is impaired in cellular model of Smith-Lemli-Opitz Syndrome

The Smith-Lemli-Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin(1A) receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin(1A) receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin(1A) receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin(1A) receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin(1A) receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.     

Ion-current-based Proteomic Profiling of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ∼40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.Smith-Lemli-Opitz syndrome (SLOS)¹ is an autosomal recessive disorder associated with subnormal growth and failure to thrive, mental retardation and neurosensory deficits, and multiple congenital anomalies, including dysmorphologies (1, 2). Early epidemiological studies estimated the incidence of SLOS as 1 in 20,000 to 1 in 60,000 live births, primarily among Caucasians (1, 2). However, more recent studies suggest that the SLOS carrier frequency is ∼1 in 30 to 1 in 50; this predicts a much higher actual disease frequency, ranging from 1 in 1,590 to 1 in 17,000 (3, 4), making SLOS the fourth most common autosomal recessive human disease (after cystic fibrosis, phenylketonuria, and hemochromatosis). Mutation of the DHCR7 gene is the intrinsic cause of SLOS; this gene encodes the enzyme DHCR7 (3β-hydroxysterol-Δ7-reductase, a.k.a. 7-dehydrocholesterol reductase; EC1.3.1.21), which catalyzes the final step in the cholesterol biosynthetic pathway, reducing the Δ7 double bond and thus converting 7-dehydrocholesterol (7DHC) to cholesterol (4, 5). As a consequence, markedly reduced levels of cholesterol and aberrantly elevated levels of the cholesterol precursor 7DHC (and its epimer, 8DHC) are observed in the majority of affected SLOS patients (6, 7). Therefore, the clinical suspicion of SLOS is confirmed by elevated 7DHC in plasma or tissues, typically demonstrated via chromatographic methods (e.g. HPLC or GC/MS) (8, 9).Visual capacity may become compromised in SLOS patients because of a variety of congenital or postnatal pathologies, such as cataracts, aniridia, corneal endothelium defects, sclerocornea, electrophysiological defects in the retina, optic nerve abnormalities, or other ophthalmologic problems (10, 11). We currently lack full knowledge of the exact pathobiological mechanism underlying SLOS, but additional insights may be afforded by studies employing a rodent model of the disease in which rats are treated with AY9944 (trans-1,4-bis[2-chlorobenzylaminomethyl] cyclohexane dihydrochloride), a relatively selective inhibitor of DHCR7 (12–14). We previously described progressive retinal degeneration in this rat model of SLOS, which is characterized by the shortening of retinal rod outer segments, pyknosis and thinning of the outer nuclear layer (ONL) of the retina (which contains the photoreceptor nuclei), and accumulation of membranous/lipid inclusions in the retinal pigment epithelium (RPE) (12, 13). Reduced rod outer segment membrane fluidity, primarily caused by a dramatic (30 to 40 mol%) decline in docosahexaenoic acid (22:6, n3) levels relative to age-matched controls, also was observed in the SLOS rat model by three postnatal months (15, 16). Retinal function and sterol steady-state in the same rat model of SLOS can be partially rescued using a high-cholesterol diet (2% by weight), although histological degeneration of the retina still occurs (17). However, the molecular mechanisms that underlie the observed electrophysiological defects in the retina, the accumulation of membranous/lipid inclusions in the RPE, the shortening of retinal rod outer segments, and the initiation of ONL pyknosis in the SLOS rat model remain poorly understood. Therefore, a comprehensive profiling of the retinal proteomes of AY9944-treated versus age-matched untreated control rats may contribute to further understanding of the underlying mechanisms responsible for the retinopathy associated with the SLOS model and, by extension, the human disease.Nevertheless, extensive and reliable expression profiling of the retinal proteome remains a prominent challenge, owing to the need to quantify data from multiple animals and a high percentage of integral membrane and membrane-associated proteins (18, 19). Label-free approaches can compare multiple replicates (20–22) with quantitative accuracy comparable to that attained with stable isotope-labeling methods (23–25). However, in order to achieve reliable relative quantification, highly quantitative and reproducible sample preparation and LC/MS analysis are required for relatively large-scale sample cohorts.In the present study, we performed a reproducible, well-controlled, ion-current-based comparative proteomic analysis of the retinas from AY9944-treated versus age/sex-matched control rats (n = 5 animals per group). A high-concentration detergent mixture was used for the efficient extraction of proteins from retinas, and samples then underwent a reproducible precipitation/on-pellet-digestion procedure and long-column, 7-h nano-LC-MS analysis. These approaches ensured extensive comparative analysis of retina samples with 10 animals. The preparative and analytical procedures were carefully optimized and controlled to ensure optimal reproducibility. Two label-free approaches, the ion-current-based method and a spectral counting method, were compared in parallel. The altered proteins were subjected to functional annotation, and selected groups of proteins of interest were further validated by means of Western blot and correlative immunohistochemical analysis.     

Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.