Abnormal gangliosides are localized in lipid rafts in Sanfilippo (MPS3a) mouse brain

Abstract

Allogenic stem cell transplantation can reduce lysosomal storage of heparan sulfate-derived oligosaccharides by up to 27 % in Sanfilippo MPS3a brain, but does not reduce the abnormal storage of sialolactosylceramide (G(M3)) or improve neurological symptoms, suggesting that ganglioside storage is in a non-lysosomal compartment. To investigate this further we isolated the Triton X100-insoluble at 4 °C, lipid raft (LR) fraction from a sucrose-density gradient from cerebral hemispheres of a 7 month old mouse model of Sanfilippo MPS3a and age-matched control mouse brain. HPLC/MS/MS analysis revealed the expected enrichment of normal complex gangliosides, ceramides, galatosylceramides and sphingomyelin enrichment in this LR fraction. The abnormal HS-derived oligosaccharide storage material was in the Triton X100-soluble at 4 °C fractions (8-12),whereas both GM3 and sialo[GalNAc]lactosylceramide (GM2) were found exclusively in the LR fraction (fractions 3 and 4) and were >90 % C18:0 fatty acid, suggesting a neuronal origin. Further analysis also revealed a >threefold increase in the late-endosome marker bis (monoacylglycerol) phosphate (>70 % as C22:6/22:6-BMP) in non-LR fractions 8-12 whereas different forms of the proposed BMP precursor, phosphatidylglycerol (PG) were in both LR and non-LR fractions and were less elevated in MPS3a brain. Thus heparan sulfate-derived oligosaccharide storage is associated with abnormal lipid accumulation in both lysosomal (BMP) and non-lysosomal (GM3 and GM2) compartments.

Fig. 1

Lipid raft isolation. a Lipid raft isolation showing protein distribution on the sucrose density gradient from a representative MPS3a brain Triton X-100 extraction. b Representative Western blot of mouse brain lipid raft preparation as shown above and isolated as described in the text. Each fraction was assayed for marker Flotillin-1 content and quantification showed that >95 % of Flotillin is in Fraction #4 (the Lipid Raft fraction)

Fig. 2

HPLC/MS/MS analysis of GM1 molecular species in Control versus MPS3a brain extracted with Triton X-100 and fractionated on a sucrose density gradient as described in the text. a GM1 containing C_18:1 sphingosine and C_18:0 fatty acid is predominantly in Fraction #4 (lipid raft) in both Control and MPS3a brain. b GM1 containing C_20:1 sphingosine and C_18:0 fatty acid is predominantly in Fraction #4 (lipid raft) in both Control and MPS3a brain. Data shown is average of analyses of cerebral cortex from two different mice

Fig. 3

HPLC/MS/MS analysis of G_M1, G_D1 and G_T1 molecular species in the lipid raft Fraction (#4) in Control versus MPS3a brain, extracted with Triton X-100 and fractionated on a sucrose density gradient as described in the text. >95 % of the GM1, GD1 and GT1 were in Fraction #4. The technique does not distinguish between individual disialoganglioses, G_D1a and G_D1b etc. or trisialogangliosides. No significant differences were observed. Data shown is average of analyses of cerebral cortex from two different control and MPS3a mice

Fig. 4

HPLC/MS/MS analysis of G_M3 molecular species in Control versus MPS3a brain extracted with Triton X-100 and fractionated on a sucrose density gradient as described in the text. a Most of the G_M3 in MPS3a brain is in lipid rafts (Fraction #4) with only trace amounts of G_M3 in controls. b Molecular species of G_M3 in Fraction #4, showing the relative amounts of C_18:1 sphingosine acylated by C_16:0, C_18:0, C_22:0 and C_24:0 fatty acids. Data shown is average of analyses of cerebral cortex from two different control and MPS3a mice

Fig. 5

Molecular species of G_M2 in Fraction #4, showing the relative amounts of C_18:1 sphingosine acylated by C_16:0 and C_18:0, fatty acids and C_20:1 sphingosine acylated by C_18:0 fatty acid (20/18) and its accumulation in MPS3a brain. Data shown is average of analyses of cerebral cortex from two different control (open bar) and MPS3a (filled bar) mice

Fig. 6

Most sphingolipids are found in Lipid Rafts (LRs). HPLC/MS/MS analysis of the major sphingolipid molecular species in Control (open bar) versus MPS3a cerebral cortex (filled bar) extracted with Triton X-100 and fractionated on a sucrose density gradient as described in the text. Results are shown for all 12 fractions with the lipid rafts being mainly in Fr #4. a C_18:1/C_24:1-ceramide. b C_18:1/C_24:1 Glyceramide. c C_18:1/C_18:0-sphingomyelin d C_18:1/C_22:0-a minor (<5 %) sphingomyelin showing presence of 85 % in non-lipid raft fractions of the sucrose-density gradient. Data shown is average of analyses of cerebral cortex from two different control and MPS3a mice

Fig. 7

HPLC/MS/MS analysis of the two major heparan sulfate-derived storage oligosaccharides in Control versus MPS3a brain extracted with Triton X-100 and fractionated on a sucrose density gradient as described in the text. Both oligosaccharides are completely absent from control brain. a GlcNS-UA) Disaccharide. Data shown is average of analyses of cerebral cortex from control (open bar) and MPS3a (filled bar) mice. b [GlcNS-UA]₂ Tetrasaccharide

Fig. 8

HPLC/MS/MS analysis of major molecular species of BMP (lyso-bis-phosphatidic acid, a–e) and its precursor PG (phosphatidylglycerol) in Control versus MPS3a brain extracted with Triton X-100 and fractionated on a sucrose density gradient as described in the text. Data shown is average of analyses of cerebral cortex from two different control (open bar) and MPS3a (filled bar) mice. a BMP containing C_22:6/C_22:6 fatty acids is increased in MPS3a brain but mostly absent from Lipid Rafts. b BMP containing C_20:4/C_22:6 fatty acids is increased in MPS3a brain but mostly absent from Lipid Rafts. c BMP containing C_18:1/C_22:6 fatty acids is increased in MPS3a brain but mostly absent from Lipid Rafts. d BMP containing C_18:1/C_20:4 fatty acids is increased in MPS3a brain but mostly absent from Lipid Rafts. e BMP containing C_18:1/C_18:1 fatty acids is increased in MPS3a brain but mostly absent from Lipid Rafts. f PG containing C_16:0/C_16:0 fatty acids (the proposed precursor of BMP) is not increased in MPS3a brain and is mainly present in Lipid Rafts. g PG containing C_18:1/C_16:0 fatty acids (34:1)(the proposed precursor of BMP) is not increased in MPS3a brain and is mainly present in Lipid Rafts. h PG containing C_18:1/C_20:4 fatty acids (38:5)(the proposed precursor of BMP) is increased in MPS3a brain and is mainly present in non-Lipid Raft fractions