Genetic Disorders Of Cholesterol Biosynthesis-Pathophysiological Studies Of Smith-Lemli-Opitz Syndrome

BackgroundCholesterol is an essential lipid in all mammalian cells,and is a major component of cell membranes.In addition to the structural role in cellular membranes,cholesterol is also the precursor molecule for sterol-based compounds including bile acids, oxysterols,neurosteroids,glucocorticoids,mineralocorticoids,and sex steroids. Moreover,the recognition that increased cholesterol level(hypercholesterolemia) is a major risk factor for the development of heart disease and atherosclerosis has gained enormous attention not only in medicine,medical and pharmacological research,but also from the general public.The discovery of a crucial role of cholesterol in human embryogenesis and the recent identification of a number of inherited disorders of cholesterol biosynthesis also show that low cholesterol level(hypocholesterolemia) may have severe consequences for human health and development.To date,nine inherited disorders of cholesterol biosynthesis have been described.Two disorders have been linked to the enzyme defects in the pre-squalene segment of the pathway: classical mevalonic aciduria and hyper-IgD and periodic fever syndrome.Remaining seven are associated with the enzymatic defects of post-squalene pathway: Smith-Lemli-Opitz syndrome(SLOS),Desmosterolosis,Hydrops-ectopic calcification-moth-eaten(HEM) skeletal dysplasia,Lathosterolosis,Antley-Bixler syndrome,Chondrodysplasia punctata 2(CDPX2),Congenital hemidysplasia ichthyosiform nevous and limb defects(CHILD) syndrome.Patients with these disorders are characterized by multiple congenital anomalies including internal organ, skeletal and/or skin abnormalities.SLOS is the first described and by far the most common disorder of post-squalene cholesterol biosynthesis.SLOS is an autosomal recessive multiple congenital anomaly/mental retardation disorder caused by enzymatic deficiency of 3β-hydroxysterol-Δ7reductase(DHCR7),an enzyme in the terminal enzymatic step of cholesterol biosynthesis.DHCR7 catalyzes both the reduction of 7-dehydrocholesterol(7DHC) to cholesterol and the reduction of 7-dehydrodesmosterol to desmosterol.Desmosterol can be converted to cholesterol by 3β-hydroxysterol-Δ24 reductase(DHCR24).Thus SLOS patients usually have low plasma cholesterol levels and invariably elevated levels of cholesterol precursors such as 7-dehydrocholesterol(and its spontaneous isomer 8-dehydrocholesterol) and absent desmosterol.The incidence of SLOS is estimated to range from 1/10,000 to 1/60,000 of live birth.Population screens for mutant DHCR7 alleles suggest a 3—4%carrier frequency in Caucasian populations,giving a hypothetical birth incidence about 1/2500-1/4500,assuming no fetal loss.Patients with SLOS display a large and variable spectrum of morphogenic and congenital anomalies,including dysmorphic craniofacial features,microcephaly,multiple internal organ,limb/skeletal and urigenital malformations,(intrauterine) growth and mental retardation,and behavioral problems.SLOS is now diagnosed by increased blood levels of 7-DHC and low cholesterol levels combination with the clinical features.There is no known cure for SLOS.Most of treatments are supportive such as surgical repair of physical anomalies of congenital heart defects,cleft palate,genital anomalies,craniofacial, gastrointestinal and limb defects.To date,a total of 121 mutations in DHCR7 have been identified Mutations are located throughout the coding region(exons 3-9),including 105 missense,five nonsense,three splice-site and eight nucleotide insertions or deletions.Fifty percent of the missense mutations are located in one of the nine predicted transmembrane domains.Based on those,to further characterize the pathophysiology and neurophysiology underlying SLOS and to provide a model system for testing therapeutic intervention,two genetically manipulated murine models by disruption of the mouse Dhcr7 gene have been generated.Dhcr7^-/- pups mimic the biochemical and phenotypic hallmarks of the SLOS. Although the genetic defects and biochemical consequences in the genetic disorders of cholesterol biosynthesis have now been identified,the pathophysiology underlying the neurodevelopmental abnormalities is poorly understood.Currently,it is not known why defects in cholesterol synthesis cause neurodevelopmental defects but a number of hypotheses have been proposed.The most popular theory is that the key morphogen,Sonic Hedgehog(Shh and its related proteins Indian and Desert Hedgehog),is affected since this protein needs covalently attached cholesterol to regulate developmental signaling processes.However,features that have been shown to be caused by defective Shh signaling in the mouse embryo are absent in Dhcr7^-/- mice.Furthermore,Shh post-translational autoprocessing and expression in brain and lung in Dhcr7^-/- embryos are not abnormal,validating the previous conclusions that precursor sterols participate as efficiently as does cholesterol in the Shh processing reaction and can also substitute for cholesterol for structural requirements such as incorporation into bilayer membranes.Thus,the data collected indicate that the pathogenic defects in cholesterol biosynthesis disorders can not be explained,at least solely at the level of disturbed hedgehog signaling cascades.We have generated transgenic mice expressing human DHCR7 cDNA(hDHCR7) driven by a brain-specific nestin promoter.Though small and subtle changes in brain sterol metabolism,we still got a stochastically partial rescue(12%) and found a delayed postnatal myelination and defective neurogenesis from the rescued mutant animals,indicating that normal central nervous system(CNS) cholesterol synthesis is critical for postnatal survival.PartⅠ.Pathophysiological consequences of transgenic reconstitution of liver-specific cholesterol biosynthesis in Dhcr7 null mouseObjectiveTargeted disruption of the murine 3β-hydroxysterol-Δ7-reductase gene(Dhcr7),an animal model of Smith-Lemli-Opitz syndrome(SLOS),leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis.To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology,we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 in the liver.MethodWe first constructed a plasmid with ApoE promoter and human DHCR7 for microinjection.Transgenic mice identified by PCR and Southern blot were called founders.The expression of human DHCR7 was detected in different tissues by RT-PCR,western blot and immunohistology.We can get transgenic Dhcr7^+/- mice (Dhcr7^+/-tg+) by mating founder with Dhcr7^+/- mouse.Dhcr7^-/-/tg+ mice were generated by mating between Dhcr7^+/-tg+ mice.To examine the changes in the transgenic mouse we used GC-MS to measure the concentration of cholesterol and 7DHC of plasma,liver,lung and brain.Lipid rafts were extracted using the classic 1% Trion X-100 to investigate the possible mechanism of cholesterol deficiency.Results1.Generation of liver-specific hDHCR7 transgenic mouseWe generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E(ApoE) promoter and its associated liver-specific enhancer.Three mice(one female and two males),referred as to TgDHCR7-1,TgDHCR7-2 and TgDHCR7-3,respectively,were identified as transgenic on the basis of PCR and Southern blot analyses of tail samples of genomic DNA.These mice were then crossed with Dhcr7^+/- mutants to generate Dhcr7^-/- mice bearing a human DHCR7 transgene.2.Liver-specific expression of hDHCR7 transgeneMultiple tissues were collected from N2 transgenic progeny from the founder mice crossed with C57B1/6J at postnatal day(P) 10 and analyzed for transgene expression by RT-PCR and immunoblotting.Both TgDHCR7-2 and TgDHCR7-3 transgenic lines expressed human DHCR7 mRNA robustly in the postnatal livers,but not in the other tissues and western blotting analyses showed expression of HA-tagged protein. TgDHCR7 mRNA transcript was detected in transgenic liver at E11.5(the earliest we could dissect liver tissue) by RT-PCR determination and was quantitatively～5 fold higher than endogenous murine Dhcr7 mRNA in the transgenic liver at E16.5 as judged by real-time PCR.Immunofluorescence staining with anti-HA antibody showed that TgDHCR7 was highly expressed in hepatocytes in liver sections,but such signal was not detectable in control liver.3.Pathophysiological effects of transgene expressionTo determine the physiological consequences of selective restoration of DHCR7 expression in liver,we bred the transgene onto Dhcr7 null background.Sterol metabolic profile,late gestational lung development,and postnatal survivability were evaluated and compared between transgenic and non-transgenic Dhcr7 knockout mice.(1) Sterol analysis:Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80～90%of normal levels in liver and lung.However,cholesterol deficiency in brain was not altered.These data indicated that selective reconstitution of DHCR7 expression in liver improved significantly cholesterol homeostasis in liver,lung and circulation of Dhcr7 null animals during embryogenesis,but did not affect metabolism in the brain.(2) Late gestational lung development:lungs from Dhcr7 null mice expressing DHCR7 in the liver showed an improved sac space formation and thinning of the prealveolar septae by histopathology,compared with non-transgenic mutant lungs. The transgene expression led to an improvement in T1-α,PECAM-1 and caveolin-1 of immunohistochemistry of lung.These all suggested that transgenic-induced improvement of cholesterol homeostasis in non-brain tissues normalized the architecture of the distal lung sacculation.(3) Postnatal survivability of Dhcr7^-/-Tg+ mice:one hundred and ninety-six pups from Dhcr7^+/-Tg+ females bred with Dhcr7^+/-Tg+ males were scored for viability.20 Dhcr7^-/-Tg+ survived the first postnatal day,but died within 48 h after birth. Lethality was minimally delayed.(4) Association of cholesterol and 7/8DHC with lipid membranes of lung tissues: cholesterol and precursor sterols were analyzed in the raft and non-raft fractions isolated by sucrose-gradient ultracentrifugation.In WT lungs,cholesterol was detected as the only major sterol,and showed a bimodal profile,with one sharp peak corresponding to the raft fractions,the second peak in high-density non-raft fractions, 7/8DHC levels were undetectable,In Dhcr7^-/-Tg- lungs,sterols composed of a mixture of 7/8DHC and cholesterol,also exhibited bimodal profiles,and 7/8DHC constituted the major sterol in both raft and non-raft fractions.In Dhcr7-/-Tg+ lungs,although cholesterol was dramatically increased in both raft and non-raft fractions, considerable amount of 7/8DHC was still detected.ConclusionThe reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues,but failed to rescue the neonatal lethality of Dhcr7 null mice.These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice,with the peripheral organs contributing the morbidity.PartⅡQuantitative proteomic analysis of brain lipid rafts in SLOS mouse modelObjectiveCentral nervous system(CNS) defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of defective cholesterol synthesis(Dhcr7^-/-) mice. Development of the brain involves many complex cholesterol-regulated events,such as membrane trafficking,signal transduction,myelin formation,and synaptogenesis. In all these developmental events,cell surface properties,determined by membrane components,are of primary importance.Deviations from normal sterol concentrations and compositions can perturb the feature of specialized lipid membrane domains (lipid rafts) and compromise cellular functions.We postulated that alterations in membrane lipid raft domains especial in cholesterol-deficient brain tissue underlay much of the pathophysiology of SLOS and determines the progression of the disease. However,the identity of possible signaling proteins had not yet been fully determined. Therefore,the objective of this study is to determine the alterations of raft protein composition in Dhcr7^-/- brains by quantitative proteomics approaches and identify the potential disease-associated biomarkers.To achieve the goals,we have developed a new method to isolate lipid rafts by detergent-free preparation,and established a tube-gel protein digestion combined with label-free tandem MS/MS(shotgun proteomics) to quantitatively analyze raft proteome.We have applied these methods to analyze the alterations of protein compositions in the lipid rafts isolated from the brains of SLOS mouse model.Methods1.Preparation of raft-enriched membranes from neonatal mouse brain by a newly devised detergent-free discontinuous Optiprep-sucrose gradients.Neonatal(postnatal day 1) mouse brains from wild-type(WT) and mutant (Dhcr7-/-) animals were used for preparation of raft-enriched membranes(lipid rafts). Cellular membranes including plasma membrane and intracellular organelle membranes were initially isolated from the crude postnuclear supernatants in 5%-35%-45%discontinuous Optiprep gradients,and they were defined to lipid membranes(LM).Subsequently,lipid rafts were purified from LM in absence of any detergent by 5%-25%-30%-35%-40%discontinuous sucrose gradients.2.Quantifying raft proteins by 'tube-gel' protein digestion label-free shotgun proteomics.The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins.We have devised a simple protocol using a 'tube-gel' protein digestion that,when combined with mass spectrometry,can be used to obtain comprehensive and reproducible identification and quantitation of the lipid raft proteome prepared from neonatal mouse brain.Lipid rafts prepared from neonatal mouse brain were directly incorporated into a polyacrylamide tube-gel matrix without prior protein separation.After in-gel digestion of proteins,nanospray LC-MS/MS,was used to analyze the extracted peptides,and the resulting spectra were searched to identify the proteins present in the sample.Relative abundances of raft proteins between WT and mutant brains were compared.Results1.Establishment a new method for isolation of lipid rafts by detergent-free approach2.Development of a new approach to quantify raft proteome by a 'tube-gel' protein digestion and label-free shotgun proteomics3.Quantitative proteomic analysis revealed multiple alterations in the lipid rafts of Dhcr7-/- brainConclusionLipid raft can be isolated efficiently from neonatal brains without TX,closer to native state of lipid rafts in cell membranes.'Tube-gel' protein digestion and label-free shotgun proteomics can get comprehensive and reproducible identification and quantitation of the lipid raft proteome.Compared with WT,many membrane proteins decreased evidently in Dhcr7-/- mouse,some involved in the developmental process of neonatal brain,including signal transduction,neurotransmitter release,and membrane trafficking.