FA2H is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin

Abstract

Myelin in the mammalian nervous system has a high concentration of galactolipids [galactosylceramide (GalCer) and sulfatide] with 2-hydroxy fatty acids. We recently reported that fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is the major fatty acid 2-hydroxylase in the mouse brain. In this report, we show that FA2H also plays a major role in the formation of 2-hydroxy galactolipids in the peripheral nervous system. FA2H mRNA and FA2H activity in the neonatal rat sciatic nerve increased rapidly during developmental myelination. The contents of 2-hydroxy fatty acids were approximately 5% of total galactolipid fatty acids at 4 days of age and increased to 60% in GalCer and to 35% in sulfatides at 60 days of age. The chain length of galactolipid fatty acids also increased significantly during myelination. FA2H expression in cultured rat Schwann cells was highly increased in response to dibutyryl cyclic AMP, which stimulates Schwann cell differentiation and upregulates myelin genes, such as UDP-galactose:ceramide galactosyltransferase and protein zero. These observations indicate that FA2H is a myelination-associated gene. FA2H-directed RNA interference (RNAi) by short-hairpin RNA expression resulted in a reduction of cellular 2-hydroxy fatty acids and 2-hydroxy GalCer in D6P2T Schwannoma cells, providing direct evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the formation of 2-hydroxy galactolipids in peripheral nerve myelin. Interestingly, FA2H-directed RNAi enhanced the migration of D6P2T cells, suggesting that, in addition to their structural role in myelin, 2-hydroxy lipids may greatly influence the migratory properties of Schwann cells.

Fig. 1

FA2H expression is upregulated during developmental myelination of rat sciatic nerve. Total RNA was isolated from rat sciatic nerves and FA2H, CGT, and P₀ mRNA levels were determined by quantitative RT-PCR. Data are normalized to 18S rRNA levels. The mean and SD are shown (n=5 for P7, P15, P20, P30, and P60; n=4 for P30). The data at P4 represents a pool of sciatic nerves from 4 rats in triplicate measurements.

Fig. 2

FA2H expression in primary Schwann cells is highly upregulated upon differentiation. Schwann cells were isolated from adult rat sciatic nerve. Total RNA was isolated from proliferating cells, control (C), or cells treated with 1 mM dibutyryl cAMP (db-cAMP). FA2H, CGT, and P₀ mRNA levels were determined by quantitative RT-PCR. Data are normalized to 18S rRNA levels.

Fig. 3

Fatty acid 2-hydroxylase activity in postnatal rat sciatic nerve. Crude nerve tissue homogenates (50 μg protein) were used for fatty acid 2-hydroxylase assays. The mean and SD are shown (n=5 for P15, P20, P30, and P60; n=3 for P7; n=2 for P10). The data at P4 represents a pool of sciatic nerve from 4 animals.

Fig. 4

Fatty acid compositions of rat sciatic nerve GalCer. Total lipids were extracted from sciatic nerve. GalCer were purified by TLC and fatty acid compositions determined by GC/MS. Average and SD of 3–4 animals are shown, except at day 4, which represents a pool of sciatic nerves from 4 animals. In all samples C_18:1 and C_24:1 fatty acids were not detectable.

Fig. 5

Fatty acid compositions of rat sciatic nerve sulfatide. Total lipids were extracted from sciatic nerve. Sulfatides were purified by TLC and fatty acid compositions determined by GC/MS. Average and SD of 3–4 animals are shown, except at day 4, which represents a pool of sciatic nerves from 4 animals. In all samples C_18:1 and C_24:1 fatty acids were not detectable.

Fig. 6

Relative contents of 2-hydroxy and non-hydroxy galactolipids of postnatal rat sciatic nerve. The 2-hydroxy (filled circle) and non-hydroxy (open circle) fatty acid contents were expressed as percent total fatty acids in purified GalCer (A) and sulfatide (B).

Fig. 7

FA2H-directed RNAi reduces cellular 2-hydroxy fatty acids and 2-hydroxy GalCer. D6P2T cells were transfected with control shRNA or FA2H shRNA expression plasmids. Transfected cells were selected for puromycin resistance (1 μg/ml) for 2 weeks. (A) Total non-hydroxy and 2-hydroxy fatty acids were measured by GC/MS following hydrolysis of all cellular lipids. (B) Cells were metabolically labeled with [¹⁴C] acetate for 48 hr. Non-hydroxy and 2-hydroxy GalCer were isolated by TLC and radioactivity was measured by liquid scintillation counting. Mean and SD of triplicate samples are shown.

Fig. 8

FA2H-directed RNAi enhances D6P2T cell migration. Cells were transfected with control or FA2H siRNA and harvested after 72 hr. (A) Fatty acid 2-hydroxylase activities in whole cell lysates. The mean and range of two measurements are shown. (B) Total 2-hydroxy fatty acids were measured by GC/MS following hydrolysis of all cellular lipids. The combined quantities of the three major 2-hydroxy fatty acids (2-hydroxy C₁₆, C₁₈, C₂₄) are shown. (C) Transfected cells (50,000 per insert) were placed in cell culture inserts and allowed to migrate for 4 hr before staining. The mean and SD of triplicate cell countings are shown.