Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice

Abstract

The cholesterol, sphingolipid, and glycerophospholipid content of total brain, of detergent-resistant membranes prepared from the total brain, and of cerebellar granule cells differentiated in culture from wild type (WT) and acid sphingomyelinase knockout (ASMKO) were studied. Brains derived from 7-month-old ASMKO animals showed a fivefold higher level of sphingomyelin and a significant increase in ganglioside content, mainly because of monosialogangliosides GM3 and GM2 accumulation, while the cholesterol and glycerophospholipid content was unchanged with respect to WT animals. An increase in sphingomyelin, but not in gangliosides, was also detected in cultured cerebellar granule neurons from ASMKO mice, indicating that ganglioside accumulation is not a direct consequence of the enzyme defect. When a detergent-resistant membrane fraction was prepared from ASMKO brains, we observed that a higher detergent-to-protein ratio was needed than in WT animals. This likely reflects a reduced fluidity in restricted membrane areas because of a higher enrichment in sphingolipids in the case of ASMKO brain.

Figure 1. Pattern of endogenous lipids from WT and ASMKO brains of seven months of life

Lipids were extracted from brain lysates and subjected to phase partitioning. Cholesterol, phospholipids and gangliosides were analysed by HPTLC followed by chemical detection as described under “Materials and Methods”. Aliquots of samples corresponding to equal amounts of proteins were analysed. A, cholesterol was separated from organic phases (corresponding to 30 μg of total proteins) using hexane/ethyl acetate (3:2, by volume) and visualized spraying the TLC with anisaldehyde. B, phospholipids were separated from the organic phases (corresponding to 120 μg of total proteins). The solvent system was chloroform/methanol/acetic acid/water (30:20:2:1 by volume), and visualization was performed with a molybdate reagent. C, gangliosides were separated from dialysed aqueous phases (corresponding to 500 μg of total proteins). The solvent system was chloroform/methanol/0.2% calcium chloride (50:42:11 by volume) and visualization was performed using a p-dimethylaminobenzaldehyde reagent. Patterns are representative of those obtained in three different experiments. Lane 1, WT brain; lane 2, ASMKO brain.

Figure 2. Distribution of sphingolipids in cerebellar granule cells on different days in culture prepared from wild type (upper graph) and ASMKO mice (lower graph)

Data were obtained from the distribution of sphingolipid-associated radioactivity after labelling with [1-³H]sphingosine and corresponding to the 3^rd (black), the 4^th (dark grey), the 5^th (light grey) and the 6^th (white) DIC. Data reported in the graph are the means of three different experiments with the S.D. in the range 10–20% of the mean values. *** p<0.001

Figure 3. Distribution of cholesterol (upper graph)and glycerophospholipids (lower graph) in the organic phases obtained from cerebellar granule cells on different days in culture prepared from wild type (black) and ASMKO (grey) mice

Data are expressed as nmol/mg of proteins and are the means of three different experiments with the S.D. in the range 10–20% of the mean values.

Figure 4. Lipid and protein distribution in sucrose gradient fractions obtained from WT mouse brain homogenate containing 3mg (panel A), 1.3 mg (panel B) and 1mg of proteins (panel C) per 1 ml of Triton X-100 in lysis buffer, by ultracentrifugation

Lipids were extracted as described under “Materials and Methods”. Equivalent volumes of the organic phases, or of the aqueous phases from gradient fractions were loaded on the TLC. Cholesterol was separated in a solvent system hexane/ethyl acetate, 3:2 (v/v) and detected by spraying the TLC with anisaldehyde. Gangliosides were separated in a solvent system chloroform/methanol/0.2%CaCl2 50:42:11(v/v/v). The TLC was incubated with Clostridium perfrigens sialidase followed by incubation with conjugated-horseradish peroxidase cholera toxin B and detected by o-phenylenediamine. Phospholipids were separated in a solvent system chloroform/methanol/acetic acid/water, 30:20:2:1 (v/v/v/v) and detected by spraying the TLC with a molybdate reagent. Same quantities of gradient fractions were analysed by SDS-PAGE followed by detection by Western blotting using specific anti-Fyn, anti-Lyn, anti-PrP and anti-Akt antibodies, as indicated on the left of each panel. Patterns are representative of one DRM preparation.

Figure 5. Lipid and protein distribution in sucrose gradient fractions obtained from ASMKO mouse brain homogenate containing 3 mg (panel A), 1.3 mg (panel B) and 1mg of proteins (panel C) per 1 ml of Triton X-100 in lysis buffer, by ultracentrifugation

Legends as in figure 5.