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Lipid composition of microdomains is altered in neuronopathic Gaucher disease sheep brain and spleen
Molecular Genetics and Metabolism, In Press, Corrected Proof, Available online 17 May 2017, Available online 17 May 2017
Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase activity that leads to accumulation of glucosylceramide and glucosylsphingosine. Membrane raft microdomains are discrete, highly organized microdomains with a unique lipid composition that provide the necessary environment for specific protein-lipid and protein-protein interactions to take place. In this study we purified detergent resistant membranes (DRM; membrane rafts) from the occipital cortex and spleen from sheep affected with acute neuronopathic Gaucher disease and wild-type controls. We observed significant increases in the concentrations of glucosylceramide, hexosylsphingosine, BMP and gangliosides and decreases in the percentage of cholesterol and phosphatidylcholine leading to an altered DRM composition. Altered sphingolipid/cholesterol homeostasis would dramatically disrupt DRM architecture making them less ordered and more fluid. In addition, significant changes in the length and degree of lipid saturation within the DRM microdomains in the Gaucher brain were also observed. As these DRM microdomains are involved in many cellular events, an imbalance or disruption of the cell membrane homeostasis may impair normal cell function. This disruption of membrane raft microdomains and imbalance within the environment of cellular membranes of neuronal cells may be a key factor in initiating a cascade process leading to neurodegeneration.
- Increases in GlcCer, HexSph, BMP and gangliosides concentrations were observed in rafts from neuronopathic Gaucher sheep.
- Decreases in the percentage of cholesterol and phosphatidylcholine were observed in rafts from neuronopathic Gaucher sheep.
- Altered sphingolipid/cholesterol homeostasis may disrupt raft architecture that in turn may impair normal cell function.
- Significant changes in the length and degree of lipid saturation were observed within rafts of neuronopathic Gaucher brain.
Abbreviations: BMP - bis(monoacylglycero)phosphate, Cer - ceramide, DRM - detergent resistant membranes, DSM - detergent soluble membranes, DHC - dihexosylceramide, GalCer - galactosylceramide, GCase - β-glucocerebrosidase, GlcCer - glucosylceramide, GlcSph - glucosylsphingosine, GD1 - disialoganglioside, GT1 - trisialoganglioside 1, GT3 - trisialoganglioside 3, GM1 - monosialotetrahexosylganglioside, GM2 - GalNAcβ4(Neu5Acα3)Galβ4GlcCer, GM3 - Neu5Acα3Galβ4GlcCer, GT1 - (GT1a, Neu5Acα8Neu5Acα3Galβ3GalNAcβ4(Neu5Acα3Galβ4GlcCer, GT1b - Neu5Acα3Galβ3GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4GlcCer, GT1c - Galβ3GalNAcβ4(Neu5Acα8Neu5Acα8Neu5Acα3)Galβ4GlcCer), GT3 - Neu5Acα2,8Neu5Acα2,8Neu5Acα2, 3Galβ1,4Glcβ1Cer (II3(NeuAc) LacCer) [trisialosyllactosylceramide], HexSph - hexosylsphingosine, MBS - MES-buffered saline, PC - phosphatidylcholine, PE - phosphatidylethanolamine, PG - phosphatidylglycerol, PI - phosphatidylinositol, SM - sphingomyelin, THC - trihexosylceramide.
Keywords: Membrane rafts, Brain lipids, Gaucher disease, Sphingolipids, Gangliosides, Glucosylceramide.
Cellular membranes are complex structures of lipids and proteins, where interactions among different components are responsible for specific cell functions as well as serving a structural purpose. Membrane lipids and proteins are not randomly distributed, but instead are organized within specific domains. Cholesterol together with glycosphingolipids and proteins are organized into specialized membrane microdomains that are called ‘lipid rafts’ or ‘membrane rafts’  and . These membrane rafts are highly dynamic assemblies that are laterally mobile, floating freely within the liquid bilayer of cellular membranes but can also cluster to form larger, ordered platforms . The molecular order of membranes, fluidity, and organization of membrane rafts are important for various cellular events and functions  and , including trafficking of membrane proteins, exo/endocytosis, cell-cell communication, signal transduction pathways , host–microbial pathogen interactions , immune recognition and intracellular vesicle trafficking . Without the correct lipid environment, proteins do not function properly; hence maintenance of lipid homeostasis is increasingly recognized as a crucial factor for normal cell function.
The ability of glycosphingolipids to act as both hydrogen bond donors and acceptors, enables their interaction with other lipids and proteins to form membrane rafts with varying architecture and function in the same cell  and . The ceramide moiety of the GSL also gives rise to heterogeneity within the membrane rafts, influencing interactions with proteins through their ceramide moiety such that interaction specificity is conferred by ceramide composition  and also influencing lipid environment and architecture , , and . Thus showing the emergence of glycosphingolipids as key regulators with membrane rafts to control cellular events, and that their deregulation may have a role in diseases such as cancers and neurodegeneration .
Membrane rafts are highly ordered and more tightly packed than non-raft domains due to intermolecular hydrogen bonding involving the saturated fatty acid side chains of the sphingolipids and cholesterol . The tight interaction between cholesterol and sphingolipids results in these liquid-ordered domains being resistant to solubilisation with detergents , and are therefore also referred to as detergent-resistant membranes (DRM). Their characteristic trait of being insoluble in nonionic detergents  as well as their low buoyant density, is used to isolate membrane microdomains, allowing analysis and characterization of the lipid composition of the DRM domains and the detergent-soluble membrane (DSM) domains in cells.
Alterations to the lipid composition of cellular membranes, in particular changes in DRM organization, have been implicated with neurodegenerative diseases  and . Alterations in the ganglioside and/or cholesterol content of DRM microdomains have been associated with Alzheimer's, Parkinson's and, Huntington's diseases, amyotrophic lateral sclerosis and prion disease , , , , and . It is hypothesized that changes in, and disruption of, the DRM environment, contributes to the loss of neural function seen in these diseases . To understand the organization and structural specificities of glycosphingolipids within membrane rafts, as well as the significance of interactions between glycosphingolipids and surrounding molecules, it is important to elucidate the physiological functions of glycosphingolipid-enriched membrane rafts and their related diseases.
Gaucher disease arises from mutations in the β-glucocerebrosidase gene which encodes the lysosomal enzyme β-glucocerebrosidase (GCase; acid β-glucosidase) . GCase mediates the hydrolysis of glucosylceramide (GlcCer) to ceramide (Cer) and glucose within the lysosome , , , and . A deficiency of GCase activity leads to lysosomal accumulation of GlcCer and its deacylated form glucosylsphingosine (GlcSph) primarily in tissue macrophages but also in other cells including neurons . We have identified a naturally occurring mutation in the β-glucocerebrosidase gene in sheep that leads to acute neurological symptoms  and .
We reported initial characterization of this sheep model of Gaucher disease and demonstrated that reduced β-glucocerebrosidase activity (1–5% of wild-type) resulted in accumulation of GlcCer and hexoylsphingosine (HexSph), as well as secondary accumulation of bis(monoacylglycero) phosphate (BMP) and gangliosides (GM1, GM2, GM3), in the brain, liver and spleen . Affected animals display an ichthyotic cutaneous appearance, akin to the collodian variant of lethal neonatal Gaucher disease in humans. Key clinical features of acute neuronopathic disease were evident. Characteristic saccadic impairment and extra ocular ophthalmoparesis were present, and motor examination confirmed a pattern of axial hypotonia and appendicular hypertonia of a spastic quality; hyper-reflexic myotatic reflexes and forelimb clonus were elicited. The animal could not support its body weight against gravity, and when positioned supine failed to right itself. Wild-type and heterozygous animals were normal on neurological examination and proved ambulant within 1 h of birth .
In this report, we examine the lipid composition of cell membrane domains in the occipital cortex of the brain and spleen of wild-type and Gaucher sheep. Preliminary analysis of total cell lipids  indicated that the occipital cortex was one of the most changed areas in the Gaucher brain, particularly the ratio of the different GlcCer species. In addition the spleen also showed a high level of GlcCer accumulation. Consequently, in this study we isolated DRM and DSM from occipital cortex and spleen tissue in the Gaucher sheep to assess whether lysosomal GlcCer accumulation alters membrane microdomain composition. This is the first study to examine the composition of DRM and DSM microdomains from Gaucher brain tissue.
2. Materials and methods
Anti flotillin-1 (polyclonal) was purchased from Sigma (St. Louis, MO: Sigma-Aldrich Cat# F1180 Lot# RRID:AB_1078893). The WestFemto ECL blotting system and Micro BCA protein assay kit were purchased from Thermo Scientific (IL, USA). HRP-conjugated goat anti-rabbit immunoglobulin was purchased from Merck Millipore (Vic, Australia: Millipore Cat# AP307P Lot# RRID:AB_92641). The internal standards Cer 18:1/17:0 [N-heptadecanoyl-d-erythro-sphingosine], BMP 14:0/14:0 [bis(monomyristoylglycero)phosphate (S,R isomer) (ammonium salt)], phosphatidylcholine (PC) 13:0/13:0 [1,2-ditridecanoyl-sn-glycero-3-phosphocholine], phosphatidylethanolamine (PE) 17:0/17:0 [1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine] and phosphatidylglycerol (PG) 14:0/14:0 [1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)] were purchased from Avanti Polar Lipids (Alabaster, AL); dihexosylceramide (DHC) 18:1/16:0 (d3) [N-palmitoyl-d3-lactosylceramide], GlcCer 18:1/16:0 (d3) [N-palmitoyl-d3-glucopsychosine], trihexosylceramide (THC) 18:1/17:0 [N-heptadecanoyl ceramide trihexoside] and GlcSph were purchased from Matreya LLC (Pleasant Gap, PA); phosphatidylcholine (PC) 14:0/14:0 [1,2-dimyristoyl-sn-glycero-3-phosphocholine] and cholesteryl heptadecanoate 17:0 were purchased from Sigma (St. Louis, MO). The d35GM1 18:1/18:0 internal standard was purchased from S, Sonnino, Department of Medical Chemistry, University of Milan, Italy. C18 solid phase extraction columns (200 mg) were obtained from UCT (Bristol, PA). All solvents were of HPLC grade, except chloroform, which contained 1% ethanol.
2.2. Tissue preparation
All animal housing, breeding and experimental procedures were approved by the SA Pathology and South Australian Health and Medical Research Institute (SAHMRI) Animal Ethics Committees in accordance with the guidelines established for the use of animals in experimental research as outlined by the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). The neuronopathic Gaucher sheep model has been previously described with wild-type control and Gaucher lambs determined by genotyping  and . The occipital cortex gray matter and spleen from three wild-type and three Gaucher newborn lambs (age-matched) were harvested, snap frozen in liquid nitrogen and stored at − 80 °C.
2.3. Isolation of DRM and DSM microdomains
DRM and DSM microdomains were extracted from the occipital cortex gray matter and spleen using the method previously described by Hattersley et al., . Briefly, tissues were cut into small pieces, placed into a glass Dounce homogeniser with 2 ml MES-buffered saline (MBS) (25 mM MES pH 6.5, 0.15 M NaCl), containing 1% (v/v) Triton X-100 and 1 mM PMSF and homogenised 20 times before being incubated on ice for 30 min. After incubation, homogenates were centrifuged at 425g for 5 min at 4 °C, after which a 50 μl aliquot of the supernatant was taken for protein determination using the Micro BCA protein assay kit. The remainder of the supernatant was put into the bottom of a Beckman (Palo Alto, CA) centrifuge tube and the sucrose concentration was adjusted to 40% (w/v) by the addition of 2 ml 80% (w/v) sucrose in MBS buffer containing 1% (v/v) Triton X-100 and 1 mM PMSF. The sample was overlayed with 5 ml 30% (w/v) sucrose in MBS buffer containing 1 mM PMSF and then 4 ml 5% (w/v) sucrose in MBS buffer containing 1 mM PMSF. Samples were centrifuged at 270,500g for 16–20 h at 4 °C in a SW40 rotor and 1 ml fractions were collected from the top of the gradient.
2.4. Western blot analysis of DRM and DSM microdomains
An aliquot of each membrane microdomain fraction (10 μl) was run on 12% SDS–PAGE gels according to the method of Laemmli . The gels were transferred to a PVDF membrane at 35 V for 70 min. The membrane was incubated in block solution (TBS + 0.1% (v/v) Tween 20 (TBST), 5% (w/v) skim milk pH 7.0) overnight at 4 °C. The following day the membrane was washed for 5 min in TBST and then incubated for 4 h at room temperature with a rabbit polyclonal antibody against flotillin-1 (diluted 1 in 1000 in block solution). The membrane was washed three times for 5 min in TBST before incubating for 1 h at room temperature in the presence of HRP-conjugated goat anti-rabbit immunoglobin (diluted 1 in 4000 in block solution). The membrane was washed again three times for 5 min in TBST before a final wash for 5 min in TBS and developed using the WestFemto ECL blotting system with detection using the LAS4000 Luminescent Image Analyser (Fujifilm Life Science, Stamford, CT, USA).
2.5. Lipid extraction from DRM and DSM microdomains
Lipids were extracted from membrane microdomain fractions (750 μl) according to the method of Bligh and Dyer . Each sample was extracted with 2.8 ml of CHCl3/CH3OH (1:2) containing 40 pmol of each of the following internal standards: GlcCer 18:1/16:0 (d3), Cer 18:1/17:0, DHC 18:1/16:0 (d3), THC 18:1/17:0, PC 14:0/14:0 (spleen only), PC 13:0/13:0 (occipital cortex only), PE 17:0/17:0, PG 14:0/14:0, BMP 14:0/14:0, cholesterol ester 17:0 and 15.8 pmol of d35GM1 18:1/18:0. An extracted 8-point calibration curve for HexSph quantification was also included, containing 0.4 to 4000 pmol of GlcSph internal standard and 40 pmol of GlcCer(18:1/16:0) (d3). The mixture was shaken for 10 min and incubated for 50 min at room temperature. Samples were partitioned by the addition of 950 μl CHCl3 and 950 μl H2O, shaken for 10 min and centrifuged at 2300g for 5 min. The upper phase containing gangliosides was collected and placed onto a C18 solid phase extraction (SPE) column that had been pre-conditioned by washing the column three times with 1 ml CH3OH followed by three times with 1 ml H2O. After loading, the SPE columns were washed with 1 ml of H2O and the gangliosides eluted into clean glass tubes with three times 1 ml CH3OH. The samples were dried under N2 at 40 °C and then reconstituted in 100 μl of CH3OH containing 10 mM NH4COOH and plated into microtitre wells for LC/ESI-MS/MS analysis. The lower hydrophobic phase was transferred to a clean tube and washed with 0.5 ml Bligh–Dyer synthetic upper phase (prepared by mixing 15 ml H2O with 56 ml CHCl3/CH3OH (1:2), shaking vigorously for 1 min, then adding 19 ml CHCl3 followed by 19 ml H2O and shaking for another min, allowing mixture to stand at room temperature overnight and retaining the top aqueous layer as the synthetic upper phase), shaken for 10 min and centrifuged at 2300g for 5 min. The upper phase was discarded and the lower hydrophobic phase was dried under a gentle stream of N2 at 40 °C. Dried lipid extracts were resuspended in 200 μl CH3OH containing 10 mM NH4COOH, centrifuged at 2300g for 5 min to remove any particulate matter and split in two and plated into microtitre wells for HexSph and BMP/PG analysis. For Cer, DHC, GlcCer (spleen only), PE, PI and THC analysis the reconstituted lipid extracts were diluted a further 1:10 in CH3OH containing 10 mM NH4COOH prior to injection. A further 1:10 dilution was done for PC and SM analysis giving a total dilution of 1:100. For GlcCer analysis of occipital cortex fractions, the dried extracts were reconstituted in 80 μl CH3CN/CH3OH/CH3COOH (97:2:1; v/v/v) containing 5 mM NH4COOH.
2.6. Quantification of BMP, Cer, DHC, GlcCer (spleen only), PC, PE, PG, PI, SM, THC and cholesterol
LC/ESI-MS/MS analysis of glycolipids and phospholipids was performed using a Shimadzu LC-20AD binary pump system combined with an AB Sciex API 4000 Q-trap triple-quadrupole mass spectrometer equipped with Analyst software (Version 1.4.2) and a turbo-ionspray source. Liquid chromatography separation of BMP, Cer, DHC, GlcCer (spleen only), PC, PE, PG, PI, SM, THC and quantification of their individual species was achieved according to Karageorgos et al. .
Following chromatography, individual species of BMP, Cer, DHC, GlcCer (spleen only), PC, PE, PG, PI and THC were quantified as previously described , with the inclusion of SM (18:0/20:0, 18:1/16:0, 18:1/16:1, 18:1/18:0, 18:1/24:0) using the m/z product ion of 184 which corresponds to the phosphocholine head group. Only the total number of carbons and double bonds of the two fatty acids are reported for the PC and PG species. PC and SM measurements from occipital cortex fractions required a different internal standard due to the presence of an interference peak. Concentrations of each molecular species were calculated by relating the peak areas of each species to the peak area of the corresponding internal standard, with PC/SM (spleen), PC/SM (occipital cortex) and PI species being related to the PC 14:0/14:0, PC 13:0/13:0 and PE 17:0/17:0 internal standards respectively, using Analyst 1.4.2 software.
Cholesterol was determined in each of the membrane microdomain fractions following lipid extraction by converting the cholesterol in each sample to cholesteryl ester by the addition of 100 μl acetyl chloride/CHCl3 (1:5; v/v) and analysed by ESI-MS/MS as described by Liebisch et al. . Relative cholesterol concentrations were determined by relating the peak area of cholesterol to the peak area of the internal standard.
2.7. Quantification of hexosylsphingosine
LC/ESI-MS/MS analysis of HexSph was determined as previously described . Using our LC/ESI-MS/MS method it was not possible to distinguish between the epimers glucosyl- and galactosylsphingosine. They did not separate chromatographically and yielded near identical MS/MS fragmentation. We therefore present overall HexSph measurements, which are the sum of the overlaying glucosyl- and galactosylsphingosine signals. Quantification was achieved from an extracted eight-point standard curve of GlcSph using the internal standard GlcCer (18:1/16:0) (d3) (m/z 865.5/264.4, retention time 6.1 min). Interpolation through the standard curve (Analyst 1.4.2 software) was performed to fit a linear regression with a 1/x2 weighting to the data based on areas for both the analyte and internal standard.
2.8. Quantification of gangliosides
LC/ESI-MS/MS analysis of gangliosides (GM1, GM2, GM3, GD1, GT1 and GT3) was performed as previously described , with the inclusion of GM1 (16:1/18:0, 18:1/18:0 and 20:1/18:0), GM3 (16:1/18:0, 18:1/18:0, 18:1/22:0, 18:1/24:0, 18:1/24:1 and 20:1/18:0), GD1 (18:1/18:0 and 18:1/20:0), GT1 (18:1/18:0, 18:1/20:0, 18:1/22:0, 18:1/24:0) and GT3 (16:1/18:0, 18:1/18:0, 18:1/22:0 and 20:1/18:0). Ganglioside concentrations were determined by relating the peak area of each species to the peak area of the d35GM1 18:1/18:0 internal standard (Analyst 1.4.2 software).
2.9. Quantification of GlcCer in occipital cortex DRM and DSM microdomains
GlcCer in the occipital cortex membrane microdomain fractions was separated away from its diastereomer galactosylceramide (GalCer) and then quantified as previously described . Concentrations of each molecular species were calculated by relating the peak areas of each species to the peak area of the internal standard GlcCer 18:1/16:0 (d3) (Analyst 1.4.2 software).
3.1. Characterization of DRM and DSM from occipital cortex and spleen of Gaucher and normal sheep
Western blot analysis with the routinely used DRM marker, Flotillin-1 , was used to localise the DRM and DSM from the occipital cortex and spleen membrane microdomain isolations (Fig. 1A, B). Fig. 1A and B shows that the majority of flotillin-1 resided in fractions four or five but was also present in fractions 10 through to 13 of the normal and Gaucher occipital cortex and spleen membrane microdomain preparations. Equal protein loading was not used as the flotillin-1 Western blot was purely performed as a profile to show separation of the DRM from the DSM. The distribution of cholesterol across all 13-membrane DRM and DSM microdomain fractions was also determined and is shown for both the normal and Gaucher occipital cortex and spleen preparations (Fig. 1C). Cholesterol was present in fractions four to six (DRM) and fractions eight to 13 (DSM). No change in cholesterol concentration was observed between wild-type and Gaucher.
Flotillin-1 and cholesterol distribution across the membrane microdomain fractions. The distribution of flotillin-1 across the 13 membrane microdomain fractions was determined by Western blotting and is shown in the occipital cortex (A) and spleen (B) from control and Gaucher lambs. The distribution of cholesterol was achieved by converting the cholesterol in each fraction to cholesteryl ester and analysis by ESI-MS/MS to determine relative cholesterol concentrations. Results are shown in nmol of cholesterol per mg of total protein loaded onto the sucrose gradient prior to fractionation (C) from the occipital cortex (squares) and spleen (triangles) in normal (solid symbols) and Gaucher (open symbols).
3.2. Distribution and increased concentration of GlcCer within membrane domains of wild-type and Gaucher occipital cortex and spleen
Total GlcCer was determined in each fraction by summing 18:1/16:0, 18:1/18:0, 18:1/20:0, 18:1/22:0, 18:1/24:0 and 18:1/24:1 species. Within wild-type occipital cortex tissue 50% (0.05 nmol/mg of protein) of total GlcCer resided in the DRM (rafts) fractions and 50% (0.05 nmol/mg of protein) in the DSM (Fig. 2A). A deficiency in GCase activity and subsequent storage of GlcCer resulted in a 140-fold and a 42-fold increase in GlcCer concentration residing in the DRM and DSM microdomains, respectively, as well as a shift in GlcCer distribution, with 75% (a mean of 6.2 nmol/mg of protein) of total GlcCer located in DRM microdomains and 25% (a mean of 2.1 nmol/mg of total protein) in the DSM microdomains of the occipital cortex in the Gaucher brain membranes (Fig. 2A). In contrast, the distribution of total GlcCer remained the same within the spleen of both Gaucher and wild-type; 65% of total GlcCer residing the DRM microdomains and 35% in the DSM microdomains – only the concentration of total GlcCer increased 12-fold in the respective domains (Fig. 2B).
Total glucosylceramide (GlcCer) in the detergent-resistant (DRM) and detergent-soluble membranes (DSM) in the occipital cortex and spleen. Concentrations of individual GlcCer species were determined across the membrane microdomain fractions using LC/ESI-MS/MS and then summed to give total GlcCer. The concentration of GlcCer in fractions four to six and fractions eight to 13 were summed to give total GlcCer in the DRM and DSM respectively from wild-type control (crosses) and Gaucher (open squares) occipital cortex (A) and spleen (B). Results are reported as nmol of GlcCer per mg of total protein loaded onto the sucrose gradient prior to fractionation.
3.3. Distribution and elevation of individual GlcCer species in occipital cortex and spleen DRM and DSM fractions from Gaucher and wild-type
In addition to a change in the distribution of total GlcCer between the DRM and DSM in the occipital cortex, the distribution of actual individual GlcCer species was also altered in Gaucher. GlcCer 18:1/18:0 was the main species in both DRM and DSM in wild-type and Gaucher occipital cortex cells while 18:1/16:0 was the main species in wild-type and Gaucher spleen cells (Fig. 3). The increase in total GlcCer in the occipital cortex was predominately due to an increase in the concentration of 18:1/18:0 and to some extent the 18:1/16:0 species. Within the DRM of occipital cortex cells, the concentration of GlcCer 18:1/18:0 increased from 0.04 nmol/mg of protein in wild-type to ~ 5.13 nmol/mg of protein in Gaucher (125-fold increase), while GlcCer 18:1/16:0 increased from 0.003 nmol/mg of protein in wild-type to 0.9 nmol/mg of protein in Gaucher (300-fold increase; Fig. 3A). In the DSM, GlcCer 18:1/18:0 increased from 0.045 nmol/mg in wild-type to 1.66 nmol/mg of protein in Gaucher (37-fold increase), while GlcCer 18:1/16:0 increased from 0.0003 nmol/mg in wild-type to 0.173 nmol/mg of protein (558-fold increase) in Gaucher (Fig. 3B).
Individual glucosylceramide (GlcCer) species in the detergent-resistant (DRM) and detergent-soluble membranes (DSM) in the occipital cortex and spleen. Concentrations of individual GlcCer species were determined across the membrane microdomain fractions using LC/ESI-MS/MS. The concentration of GlcCer in fractions four to six and fractions eight to 13 were summed to give total 18:1/16:0, 18:1/18:0, 18:1/20:0, 18:1/22:0, 18:1/24:0 and 18:1/24:1 GlcCer in the DRM (A and C) and DSM (B and D) from the occipital cortex (A and B) and spleen (C and D) in wild-type control (open bars) and Gaucher (solid bars) lambs. Results are reported as nmol of GlcCer per mg of total protein loaded onto the sucrose gradient prior to fractionation and expressed as the mean and SD (n = 3).
Significant at *P < 0.05, #P < 0.01, ^P < 0.001 (Student t-test).
Storage of GlcCer within the spleen showed a different pattern of GlcCer distribution and of species type. The individual GlcCer species increased in both the DRM and DSM (Fig. 3C, D, respectively). The major species in the spleen, GlcCer 18:1/16:0, increased 13- and 16-fold in the DRM and DSM, respectively (Fig. 3C, D). The other GlcCer species analysed (18:1/18:0, 18:1/20:0, 18:1/22:0, 18:1/24:0 and 18:1/24:1) also increased in concentration six- to 19-fold in Gaucher DRM and DSM compared to wild-type. Higher concentrations of the long chain GlcCer species (18:1/22:0, 18:1/24:0 and 18:1/24:1) were detected in both the DRM and DSM of Gaucher spleen cells than in Gaucher brain cells.
3.4. Increased HexSph concentrations within DRM and DSM fractions of occipital cortex and spleen
Extremely low concentrations of HexSph (GlcSph plus galactosylsphingosine) were detected in wild-type occipital cortex tissue (0.1 nmol/mg of protein in the DRM and 0.05 nmol/mg of protein in the DSM). HexSph concentrations increased to a mean of 35 nmol/mg of protein in the DRM from the Gaucher occipital cortex and a mean of 29 nmol/mg of protein in the DSM, corresponding to a 350-fold and 580-fold increase, respectively (Fig. 4A). Hence, within Gaucher occipital cortex tissue 55% of HexSph resided in the DRM and 45% in the DSM domains. Very low amounts of HexSph were also detected in the spleen of wild-type lambs (0.32 pmol/mg in the DRM and below the limit of detection in the DSM domains; Fig. 4B). In the Gaucher spleen however, the deficiency in GCase activity led to the storage of HexSph within both DRM and DSM domains. Within the Gaucher spleen, HexSph was stored predominately in the DSM domains with a mean of 16 nmol/mg of protein (89% of total HexSph), compared to a mean of 2 nmol/mg of protein (11% of total HexSph) in the DRM.
Hexosylsphingosine (HexSph) concentrations in the detergent-resistant (DRM) and detergent-soluble membranes (DSM) in the occipital cortex and spleen. HexSph concentrations were determined across the membrane microdomain fractions using LC/ESI-MS/MS. The concentration of HexSph in fractions four to six and fractions eight to 13 were summed to give total HexSph in the DRM and DSM respectively from wild-type control (crosses) and Gaucher (open squares) occipital cortex (A) and spleen (B). Results are reported as nmol of HexSph per mg of total protein loaded onto the sucrose gradient prior to fractionation.
3.5. Elevated BMP within the DRM and DSM fractions of spleen and occipital cortex
Total BMP was determined by summing the individual BMP species analysed (16:0/16:0, 16:1/16:0, 16:1/18:1, 18:1/16:0, 18:1/18:1, 18:1/18:2, 18:1/20:4, 18:1/22:6, 20:4/22:6, and 22:6/22:6) within the DRM and DSM fractions of occipital cortex and spleen (plus 20:4/20:4 and 22:5/22:5 for spleen). Within wild-type occipital cortex tissue the majority (98.5% of total BMP; a mean of 0.24 nmol/mg) resided in the DSM domains and only 1.5% (a mean of 0.004 nmol/mg of protein) in the DRM (Fig. 5A). In the Gaucher occipital cortex however, the consequentially accumulated and stored BMP resulted in a 46-fold increase in BMP (a mean of 0.17 nmol/mg of protein) residing in the DRM and a 5-fold increase (a mean of 1.16 nmol/mg of protein) residing in the DSM domains (Fig. 5A). The subsequent distribution of this stored BMP within the cell membranes of the Gaucher occipital cortex resulted in 12.5% of total BMP located in DRM and 87.5% in the DSM domains. The increase in total BMP recorded in the DSM of Gaucher occipital cortex was not statistically significant. However, one of the three Gaucher lambs sampled died in utero, and the BMP from this lamb was not increased to the same extent as the other two Gaucher lambs but still higher than wild-type.
Total bis(monoacylglycero)phosphate (BMP) in the detergent-resistant (DRM) and detergent-soluble membranes (DSM) in the occipital cortex and spleen. Concentrations of individual BMP species were determined across the membrane microdomain fractions using LC/ESI-MS/MS and then summed to give total BMP. The concentration of BMP in fractions four to six and fractions eight to 13 were summed to give total BMP in the DRM and DSM respectively from wild-type control (crosses) and Gaucher (open squares) occipital cortex (A) and spleen (B). Results are reported as nmol of BMP per mg of total protein loaded onto the gradient prior to fractionation.
When comparing the distribution of BMP within the wild-type spleen cells to that of brain cells, a slightly higher proportion of BMP was located in the DRM fraction (a mean of 0.01 nmol/mg of protein; 7.5% of total BMP), with a mean of 0.13 nmol/mg of protein (92.5% of total BMP) in the DSM of spleen cells (Fig. 5B). The distribution of BMP within Gaucher spleen cells (a mean of 0.19 nmol/mg BMP in the DRM and a mean of 1.33 nmol/mg BMP in the DSM; Fig. 5B) was virtually identical to that detected in the cells of Gaucher occipital cortex (12.5% in the DRM and 87.5% in the DSM).
3.6. Distribution and elevation of individual BMP species within DRM and DSM fractions of occipital cortex and spleen
The long chain unsaturated species 22:6/22:6 and 18:1/22:6, as well as 18:1/18:1 were the most abundant BMP species in cell membranes of the occipital cortex of both wild-type and Gaucher, residing predominantly in the DSM domains (Fig. 6B). Of these major BMP species, 18:1/18:1 was significantly increased in Gaucher DSM domains (200 pmol/mg of protein; 32-fold increase) compared to wild-type (6.4 pmol/mg of protein). Within the DRM of the occipital cortex, all individual BMP species were increased in Gaucher compared to wild-type, however only significant increases in BMP 16:0/16:0, 16:1/16:0, 16:1/18:1, 18:1/16:0 and 18:1/18:1 concentrations were observed (Fig. 6A).
Individual bis(monoacylglycero)phosphate (BMP) species in the detergent-resistant (DRM) and detergent-soluble membranes (DSM) in the occipital cortex and spleen. Concentrations of individual BMP species were determined across the membrane microdomain fractions using LC/ESI-MS/MS. The concentration of BMP in fractions four to six and fractions eight to 13 were summed to give totals of the individual BMP species in the DRM (A and C) and DSM (B and D) from the occipital cortex (A and B) and spleen (C and D) in wild-type control (open bars) and Gaucher (solid bars) lambs. Results are reported as pmol or nmol of BMP per mg of total protein loaded onto the gradient prior to fractionation and expressed as the mean and SD (n = 3).
Significant at *P < 0.05, #P < 0.01 (Student t-test).
The unsaturated BMP species 18:1/18:1 was the main BMP species in the Gaucher spleen with 122 pmol/mg and 914 pmol/mg of protein residing in the DRM and DSM domains, respectively (Fig. 6C, D). The long chain unsaturated species 18:1/22:6 was the next most abundant, with 126 pmol/mg of protein residing in the DSM microdomains of Gaucher spleen (Fig. 6D). Although significant increases in the concentration of most BMP species were observed within the DRM of the spleen, concentrations were low (Fig. 6C).
3.7. Increased concentration of Total GM1, GM2 and GM3 gangliosides in the DRM and DSM from occipital cortex and spleen
The concentration of total GM1/GM2/GM3 gangliosides increased from 19.8 nmol/mg of protein in wild-type to 65.4 nmol/mg of protein in Gaucher occipital cortex (data not shown). The ganglioside GM1 was the main ganglioside found in brain cells (Fig. 7A). Within the wild-type occipital cortex, 63% (10 nmol/mg of protein; Fig. 7A) of GM1 was distributed within the DRM domains and 37% (5.7 nmol/mg; Fig. 7) of GM1 was found within DSM domains. Within Gaucher occipital cortex however, the GM1 concentration was not only higher but three-fold more GM1 was located in the DRM domain (33 nmol/mg GM1 in DRM compared to 9.9 nmol/mg GM1 in DSM). GM2 and GM3 were more highly elevated in GD occipital cortex, with the majority of GM2 and GM3 residing in the DRM. A 25-fold increase of GM3 concentration was seen in the DRM of Gaucher occipital cortex (8.0 nmol/mg GM3 in Gaucher compared to 0.32 nmol/mg in wild-type) and an eight-fold increase in GM3 in the DSM domains (2.1 nmol/mg GM3 in Gaucher compared to 0.26 nmol/mg GM3 in wild-type) (Fig. 7A). A four-fold and two-fold increase in GM2 concentration was seen in the DRM and DSM domains in Gaucher occipital cortex respectively. The levels of GM1, GM2 and GM3 were considerably lower in spleen cells (< 0.1 nmol/mg of protein in wild-type and < 0.3 nmol/mg of protein in Gaucher) compared to brain cells (Fig. 7B).
Total gangliosides (GM1, GM2, GM3) in the detergent-resistant (DRM) and detergent-soluble membranes (DSM) in the occipital cortex and spleen. Concentrations of individual GM1, GM2 and GM3 species were determined across the membrane microdomain fractions using LC/ESI-MS/MS and then summed to give total GM1, GM2 and GM3. The concentration of GM1, GM2 and GM3 in fractions four to six and fractions eight to 13 were summed to give total amounts in the DRM and DSM respectively from wild-type control (open bars) and Gaucher (solid bars) occipital cortex (A) and spleen (B) samples. Results are reported as nmol of GM1, GM2 or GM3 per mg of total protein loaded onto the sucrose gradient prior to fractionation and expressed as the mean and SD (n = 3).
Significant at *P < 0.05, #P < 0.01 (Student t-test).
Concentrations of the GD1, GT1, and GT3 gangliosides were also determined in the DRM and DSM, however the concentration of these gangliosides were below the level of detection in spleen tissue and no significant differences were detected in the occipital cortex. Within the wild-type occipital cortex, 57% (0.35 nmol/mg of protein) of GD1 was distributed within the DRM domains and 43% (0.26 nmol/mg of protein) of GD1 was found within DSM domains. Similarly, 58% (0.4 nmol/mg of protein) of GD1 was distributed within the Gaucher occipital cortex DRM domains and 42% (0.3 nmol/mg of protein) of GD1 was found within DSM domains (results not shown). No significant difference was seen in the concentration and distribution of the ganglioside GT1 (0.13 nmol/mg in Gaucher occipital cortex rafts compared to 0.14 nmol/mg of protein in wild-type) occipital cortex rafts. The concentration of the GT3 ganglioside was below the level of detection.
3.8. Distribution and elevation of individual GM1, GM2 and GM3 species within DRM and DSM fractions of occipital cortex and spleen
The GM1 species 18:1/18:0 was the main species of all gangliosides in both wild-type and Gaucher occipital cortex cells and the majority resided in the DRM (Fig. 8). A significant increase (P < 0.05) in the concentration of GM1 18:1/18:0 (6.7 nmol/mg of protein in wild-type compared to 24 nmol/mg of protein in Gaucher) was seen within the DRM (Fig. 8A). Similarly all the individual GM1, GM2 and GM3 ganglioside species were also significantly increased in the DRM of Gaucher occipital cortex cells (Fig. 8A, C, E). The highest increases in concentrations were that of the GM3 species (16:1/18:0, 18:1/18:0, 18:1/22:0, 18:1/24:0, 18:1/24:1 and 20:1/18:0) within the DRM (Fig. 8E). A 125-fold increase in the long chain saturated species GM3 18:1/22:0 (0.54 pmol/mg of protein in wild-type to 67 pmol/mg of protein in Gaucher), and a 90-fold increase (0.4 pmol/mg of protein in wild-type to 36 pmol/mg of protein in Gaucher) in GM3 18:1/24:0 concentration was seen in the DRM of Gaucher occipital cortex compared to wild-type. GM3 species 16:1/18:0, 18:1/24:1 and 20:1/18:0, GM1 16:1/18:0 and GM2 16:1/18:0 were also significantly elevated within the DSM domains in Gaucher.
Individual ganglioside (GM1, GM2 and GM3) species in the detergent-resistant (DRM) and detergent-soluble membranes (DSM) in the occipital cortex. Concentrations of individual GM1, GM2 and GM3 species were determined across the membrane microdomain fractions using LC/ESI-MS/MS. The concentration of individual GM1, GM2 and GM3 species in fractions four to six and fractions eight to 13 were summed to give totals of the individual GM1 (A and B), GM2 (C and D) and GM3 (E and F) species in the DRM (A, C and E) and DSM (B, D and F) from the occipital cortex in wild-type control (open bars) and Gaucher (solid bars) lambs. Results are reported as nmol or pmol of GM1, GM2 and GM3 per mg of total protein loaded onto the sucrose gradient prior to fractionation and expressed as the mean and SD (n = 3).
Significant at *P < 0.05, #P < 0.01, ^P < 0.001 (Student t-test).
3.9. Altered lipid composition of the DRM in the occipital cortex and spleen of wild-type and Gaucher lambs
The concentrations of each lipid type (Cer, GlcCer, dihexosylceramide (DHC), trihexosylceramide (THC), BMP, phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), HexSph, GM1, GM2, GM3, cholesterol, phophatidylcholine (PC) and sphingomyelin (SM)) were determined in the DRM using LC/ESI-MS/MS. The total concentration of these lipids was summed to give total lipid concentration; each lipid type was then expressed as a percentage of total in wild-type and Gaucher (Fig. 9). In wild-type occipital cortex tissue the DRM containing fractions are made up predominantly of PC, SM, GM1, cholesterol, PE and GM2, (approximately 57%, 20%, 11%, 4.7%, 3.3% and 2.4%, respectively; Fig. 9A), whereas all other lipids each made up < 1%. In Gaucher occipital cortex tissue however, the DRM (fractions 4 to 6) is made up predominantly of PC, HexSph, GM1, SM, GM2, GM3 and GlcCer (Fig. 9A). Within the occipital cortex the percentage of GlcCer increased from 0.05% in wild-type to 3.4% in Gaucher, while HexSph increased from 0.1% in wild-type to 19% in Gaucher DRM (Fig. 9A). Similarly the percentage of GM3 increased from 0.3% in wild-type to 4.4% in Gaucher DRM of the occipital cortex. The percentage of GM2 increased from 2.4% in wild-type to 5.2% in Gaucher DRM, while the percentage of GM1 increased from 11% in wild-type to 18% in Gaucher DRM in the occipital cortex (Fig. 9A). Consequently, a decrease was seen in the other normally abundant DRM lipids (PC decreased from 57% to 34%; SM decreased from 20% to 11%) creating a completely altered DRM lipid composition.
Lipid composition of the detergent-resistant membranes (DRM) in the occipital cortex and spleen. The amount of each lipid type (Cer, GlcCer, DHC, THC, BMP, PG, PE, PI, HexSph, GM1, GM2, GM3, cholesterol, PC, SM) in the occipital cortex (A) and spleen (B) DRM were summed to give the total lipid concentration; each lipid type was then expressed as a percentage of total in wild-type control (open bars) and Gaucher disease (solid bars) lambs.
Within wild-type DRM isolated from spleen tissue, PC (34% of total DRM lipid) is the most abundant lipid, followed by SM (26%) and cholesterol (17%) (Fig. 9B). Within Gaucher spleen cells, the deficiency in GCase activity resulted in GlcCer being the most abundant lipid residing in the DRM (35% of total DRM lipid), followed by PC (19%), SM (14%) and cholesterol (10%) (Fig. 9B). The concentration of HexSph was below the level of detection in the wild-type spleen DRM fraction but was accumulating within the Gaucher DRM fraction, making up 7% of total lipids in the DRM.
3.10. Quantification of other sphingolipids, phospholipids and cholesterol
Cer, DHC, THC, SM, PC, PE, PG, PI and cholesterol concentrations were also determined in the DRM and DSM from sheep occipital cortex and spleen. No significant differences were detected in these sphingolipids, phospholipids and cholesterol apart from THC and PG in the DRM from the occipital cortex and spleen respectively, where they were elevated in Gaucher, but concentrations were very low (data not shown). There was a trend for DHC to be increased in the DRM and DSM of the occipital cortex however this was not statistically significant (data not shown).
DRM domains (lipid rafts) are associated with membrane organization, acting as dynamic platforms for cell signalling, protein processing and membrane turnover. Altered lipid composition and abnormal DRM organization and structure, disrupts DRM-dependent signalling  and . Abnormal DRM structure and function have been implicated in several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's diseases, and prion diseases , , and .
We have previously described neuronopathic Gaucher disease in lambs which present at birth with acute neurological symptoms  and . Affected lambs found deficient in glucocerebrosidase activity were shown to be homozygous for the missense mutations c.1142G > A (p.C381Y) and c.1400C > T (p.P467L) . In this study, we investigated the effect of deficient GCase activity and the impaired degradation of GlcCer on the lipid composition of DRM in the brain and spleen of newborn neuronopathic Gaucher disease lambs. GCase activity within the Gaucher occipital cortex was 0.02 nmol/min/mg of protein compared to 1 nmol/min/mg of protein in wild-type occipital cortex (2% of wild-type GCase activity ). GCase activity in the Gaucher spleen was 0.02 nmol/min/mg compared to 0.45 nmol/min/mg in wild-type spleen (4.4% of wild-type spleen ).
The overall increase in sphingolipid concentration detected in this study, presumably is occurring not only within the lysosome but also outside of the lysosome in non-lysosomal membranes. The pathological hallmark in all lysosomal storage diseases is the initial accumulation of a specific substrate inside organelles of the endosomal–lysosomal system, however in many lysosomal storage diseases accumulation of glycosphingolipids have previously been identified in other cellular membranes besides the endosomal-lysosomal system such as the endoplasmic reticulum, golgi and at the mitochondrial membrane . Accumulation of these storage compounds in endoplasmic reticulum membranes affects several functions such as Ca2 + homeostasis  and signalling cascades . Such redistribution of lipids for the endosomal/autolysosomal system to other cellular membranes could have functional implications and may actively contribute to the pathogenic cascade . Our group has previously isolated DRM from lysosomes and showed that their lipid composition was similar to that isolated from total cells in the Gaucher cell model ; however, the amounts of each of the individual lipid species were much lower in the lysosomal DRM, compared with the total cell DRM, suggesting that although there may be some contamination of the DRM from the lysosomes, it does not account for the entire altered lipid composition.
Secondary accumulation of BMP resided mainly in the DSM microdomains. Mammalian cells contain relatively low amounts of BMP, generally no > 1–2% of total phospholipids. BMP is localized in the internal membranes of late endosomes and lysosomes and is generally detected in the DSM microdomains but can also be detected in the DRM microdomains of late endosomes . Within wild-type occipital cortex tissue, 98.5% of total BMP resided in the DSM microdomains and only 1.5% in the DRM (Fig. 5A). A 46-fold increase in total BMP in the DRM and a 5-fold increase in the DSM was observed in the Gaucher occipital cortex. The increase in BMP concentration in our model implies that late endosomal or lysosomal membrane mass is increased. Separation of the plasma membrane from the lysosomes by subcellular fractionation would help clarify this, however due to logistics and the distance from our lambing/post-mortem location to our experimental laboratory, and the requirement of fresh tissue for cell fractionation, we were unable to separate the membranes in this round of experiments.
Generally PC makes up > 50% of the lipid species in mammalian membranes, however in Gaucher, PC was only 34% of total lipid making up the DRM. As seen within the Gaucher spleen membranes, GlcCer, PC SM, cholesterol, HexSph and Cer were the major lipids, however, within the Gaucher brain the gangliosides GM1, GM2, GM3, along with PC, HexSph, and SM were most abundant. Gangliosides are concentrated in the brain cell membranes and serve a number of critical cellular functions, including cell signalling, cell-cell recognition and the maintenance and repair of nervous tissues , , and . Disruption of cellular ganglioside homeostasis is implicated in central nervous system disease . In Gaucher lamb brain, gangliosides GM1, GM2, and GM3 were elevated and were also more concentrated in the DRM microdomains (Fig 7 and Fig 8).
Within the lipid bilayer of biological membranes, unsaturated glycerophospholipids are believed to be loosely-packed, forming domains that are in a liquid disordered (fluid) state. DRM microdomains consist of lipids with saturated acyl chains that are tightly packed, forming a liquid-ordered state (gel phase) , , and . DRM are more highly ordered and rigid than DSM microdomains due to the interaction between sphingolipids and cholesterol. The relative fluidity of the membrane lipid bilayer is an important factor in determining and controlling the functionality of the cell.
Recent studies provide evidence that alterations in lipid composition have major effects on the physiochemical properties of DRM microdomains, such as affecting the fluidity of the membrane. The length and degree of saturation of lipids are important determinants of membrane thickness, fluidity, local curvature, and molecular packing within the cell membrane, which in turn regulate the activities of membrane-bound enzymes . Incorporation of saturated fatty acids into membranes results in decreased membrane fluidity . We have shown in this study that the composition of Gaucher DRM are dramatically altered, with massive increases in GlcCer species with short chain fatty acid acyl chains in lieu of an equal distribution of the different GlcCer species C16-C24. Significant increases in the concentrations of both saturated GlcCer 18:1/18:0 and 18:1/16:0 species (125- and 300-fold respectively) were present in the DRM microdomains of the Gaucher occipital cortex, while concentrations of the long acyl chain GlcCer species (18:1/22:0, 18:1/24:0 and 18:1/24:1) were increased to a significantly less extent (Fig. 3A). The predominating shorter chain saturated 18:1/18:0 species in the Gaucher brain is likely to influence membrane properties such as fluidity. The long unsaturated acyl chain 18:1/24:1 GlcCer species was below the level of detection within the DRM microdomains of wild-type brain but was significantly increased in the Gaucher DRM, possibly influencing the DRM microdomain structure.
The data presented in this study support the hypothesis that alteration in the DRM microdomain environment results in less ordered DRM microdomains and less structurally-sound signalling platforms that may be important in the pathogenic pathway of neurodegenerative diseases. Altered sphingolipid/cholesterol homeostasis would result in alterations in lipid and protein ‘packaging’, due to alterations in sphingolipid and cholesterol ratios; additional changes in the saturation of the hydrocarbon chains in DRM sphingolipids and phospholipids may also result in packing defects of the DRM microdomains that in turn may impair protein function and cell signalling.
Significant increases in the concentrations of glucosylceramide, hexosylsphingosine, BMP and gangliosides and decreases in the percentage of cholesterol and phosphatidylcholine were observed in detergent resistant membranes (membrane rafts) from the occipital cortex and spleen from sheep affected with acute neuronopathic Gaucher disease and wild-type controls. Altered sphingolipid/cholesterol homeostasis would dramatically disrupt detergent resistant membrane architecture making them less ordered and more fluid that in turn may impair protein function, cell signalling and normal cell function.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
We thank Dr. Marten Snel and Stephen Duplock at the Mass Spectrometry Facility at SAHMRI for assistance with mass spectrometry analysis, as well as Dr. Tim Kuchel and the staff at the SAHMRI Large Animal Facility at Gilles Plains for their help during lambing.
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Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, Adelaide, South Australia, 5001, Australia
⁎ Corresponding author at: Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, PO Box 11060, Adelaide, South Australia, 5001, Australia.
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