Dynamics of sphingolipids and the serine palmitoyltransferase complex in rat oligodendrocytes during myelination

Abstract

Myelin is a unique lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical myelin components. Yet sphingolipid content and synthesis have not been well characterized in oligodendrocytes, the myelin-producing cells of the CNS. Here, using quantitative real-time PCR, LC-MS/MS-based lipid analysis, and biochemical assays, we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we characterized sphingolipid production in isolated oligodendrocytes. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination, levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of two alternative catalytic subunits, SPTLC2 and -3, in the relative levels of isoforms of the small subunits, ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. Myelination progression was accompanied by distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. We conclude that the distribution of these changes among sphingolipid family members is indicative of a selective channeling of the ceramide backbone toward specific downstream metabolic pathways during myelination. Our findings provide insights into myelin production in oligodendrocytes and suggest how dysregulation of the biosynthesis of this highly specialized membrane could contribute to demyelinating diseases.

Keywords: ORMDL sphingolipid biosynthesis regulator; brain lipids; ceramide; demyelinating disease; developmental myelination; glycolipids; lipidomics; sphingolipids.

Fig. 1.

Key proteins associated with myelination increase with age in the developing rat brain. A: Western blot analysis of total membranes isolated from the brains of postnatal rats of various ages as indicated in each panel. Blots were probed with primary antibodies for the myelin-associated proteins MOG, PLP, and MBP with β-actin as a loading control. B: Total RNA was isolated from the brains of postnatal rats of various ages as indicated. RT-qPCR analysis was performed and mRNA expression levels of key genes associated with myelination were quantitated. Data have been normalized to GAPDH expression levels and then set relative to day 2. Shown are mean ± SEM, n = 5 animals per age group. HPRT, hypoxanthine phosphoribosyltransferase.

Fig. 2.

Mass levels of key sphingolipids and corresponding sphingolipid metabolic enzymes associated with myelination increase in the postnatal rat brain. Total membranes were isolated from the brains of postnatal rats of various ages as indicated. Steady state levels of d18:1 monohexosylceramides (A) and d18:1 sulfatides (B) were quantitated using LC-MS/MS analysis. Data are graphed as picomoles of lipid per 100 μg of total protein. C: Total RNA was isolated from the brains of postnatal rats of various ages as indicated. RT-qPCR analysis was performed and mRNA expression levels of key enzymes responsible for the biosynthesis of monohexosylceramides and sulfatides were quantitated. Data have been normalized to GAPDH expression levels and then set relative to day 2. Data are presented as mean ± SEM, n = 5 animals per age group. Ugt8, UDP-glycosyltransferase 8.

Fig. 3.

Steady state levels of d18:1 sphingolipids in the membranes of the postnatal rat brain. Total membranes were isolated from the brains of postnatal rats of various ages as indicated in each panel. Steady state levels of total d18:1 ceramides (A), d18:1 SMs (B), d18:1 sphingosine (C), d18:1 dihydro-sphingosine (D), d18:1 S1P (E), and d18:1 dihydro-S1P (F) were quantitated using LC-MS/MS analysis. Data are graphed as picomoles of lipid per 100 μg of total protein. Data are expressed as mean ± SEM, n = 5 animals per age group.

Fig. 4.

Steady state levels of total d18:1 sphingoid base lipids were measured in the oligodendrocytes isolated from the brains of postnatal rats of various ages. LC-MS/MS was used to quantitate the mass levels of total d18:1 ceramides (CER), d18:1 SMs, d18:1 GluCERs, d18:1 GalCERs, and d18:1 sulfatides (Sulfatide) in both the primary oligodendrocytes isolated from the brain tissue of postnatal rats using the Percoll gradient method as described in the Isolation of primary oligodendrocytes from rat brain section of the Materials and Methods. Data are graphed as picomoles of lipid per 100 μg of total protein. Samples are from pooled oligodendrocytes derived from 20 rat pups.

Fig. 5.

Dynamic changes in the activity and subunit composition of SPT in the early postnatal rat brain. A: SPT activity was measured using an HPLC-ESI-MS/MS method combined with deuterated serine to quantitatively measure 3-KDS generated in membranes from postnatal rat brains. Data are graphed as picomoles of 3-KDS lipid per milligram of total protein. Data are expressed as mean ± SEM, n = 5 animals per age group. B: Western blot analysis of total membranes isolated from the brains of postnatal rats of various ages as indicated in each panel. Blots were probed with primary antibodies for the large subunits of SPT (SPTLC1-3), total ORMDL protein, and calnexin.

Fig. 6.

mRNA expression levels of the SPT large and small subunits are dynamically regulated in the developing rat brain. Total RNA was isolated from the brains of postnatal rats of various ages as indicated in each panel. RT-qPCR analysis was performed and mRNA expression levels of the large subunits, SPTLC1 (A), SPTLC2 (B), and SPTLC3 (C), as well as the small subunits, ssSPTa (D) and ssSPTb (E), were quantitated. Data have been normalized to GAPDH expression levels and then set relative to day 2. Data are presented as mean ± SEM, n = 5 animals per age group.

Fig. 7.

mRNA expression levels of the three ORMDL isoforms are dynamically regulated in the developing rat brain. Total RNA was isolated from the brains of postnatal rats of various ages as indicated in each panel. RT-qPCR analysis was performed and mRNA expression levels of ORMDL1 (A), ORMDL2 (B), and ORMDL3 (C), as well as the housekeeping gene, hypoxanthine phosphoribosyltransferase (HPRT) (D), were quantitated. Data have been normalized to GAPDH expression levels and then set relative to day 2. Data are expressed as mean ± SEM, n = 5 animals per age group.

Fig. 8.

SPT and ORMDL proteins are temporally regulated in oligodendrocytes isolated from the brains of postnatal rats of various ages. Primary oligodendrocytes were isolated by Percoll gradient purification as detailed in the Materials and Methods. After isolation, half of the cells were homogenized and used for Western blot analysis of SPT subunits and MBP as depicted in A. Calnexin is included as a loading control. The remaining cells were used for total RNA isolation and real-time qPCR analysis to quantitate the mRNA expression levels of the ORMDL isoforms (B), the SPT subunits (C), and the oligodendrocyte-specific genes (D). mRNA expression data were normalized to GAPDH expression levels and then set relative to day 2. Data are expressed as the mean ± SEM of triplicate technical replicates. Shown is one of two determinations that yielded similar results.

Fig. 9.

The incorporation of d16:1 and d20:1 sphingoid bases into sphingolipids in oligodendrocytes during myelination. Oligodendrocytes were isolated from the brains of rat pups at days 2, 8, and 16 after birth; lipids were extracted and subjected to analysis by MS. A: Levels of d16:1 sphingoid base incorporated into ceramides, monohexosylceramides, and SM. B: Levels of d20:1 sphingoid base incorporated into ceramides, monohexosylceramides, and SM. Data are graphed as percent of each species as a total of d18:1 sphingolipid. Mass levels of these samples are presented in the supplemental material. Single samples from oligodendrocytes isolated from 20 pups are shown.

Fig. 10.

The molecular species of sphingolipids change dramatically over time and differ between total brain and isolated oligodendrocytes in the postnatal rat brain. Membranes prepared from total brain (A, D, G) or isolated oligodendrocytes (B, C, E, F, H) just prior to myelination (postnatal day 2), early in myelination (day 9 for total brain, day 8 for oligodendrocytes), and during peak myelination (day 15 in brain, day 16 in oligodendrocytes) were analyzed by MS. For clarity, only the major molecular species (with some exceptions noted in the Results) are shown. Ceramides (A, B), SMs (D–E), total hexosylceramides (G), GluCers (H), GalCers (C), and sulfatides (F). For total brain n = 5; for oligodendrocytes, single samples from oligodendrocytes isolated from 20 pups are shown. For brain fractions, mean ± standard deviation is shown.