miR-32 and its target SLC45A3 regulate the lipid metabolism of oligodendrocytes and myelin

Abstract

Oligodendrocytes generate large amounts of myelin by extension of their cell membranes. Though lipid is the major component of myelin, detailed lipid metabolism in the maintenance of myelin is not understood. We reported previously that miR-32 might be involved in myelin maintenance (Shin et al., 2009). Here we demonstrate a novel role for miR-32 in oligodendrocyte function and development through the regulation of SLC45A3 (solute carrier family 45, member 3) and other downstream targets such as CLDN-11. miR-32 is highly expressed in the myelin-enriched regions of the brain and mature oligodendrocytes, and it promotes myelin protein expression. We found that miR-32 directly regulates the expression of SLC45A3 by binding to the complementary sequence on the 3'UTR of cldn11 and slc45a3. As a myelin-enriched putative sugar transporter, SLC45A3 enhances intracellular glucose levels and the synthesis of long-chain fatty acids. Therefore, overexpression of SLC45A3 triggers neutral lipid accumulation. Interestingly, both overexpression and suppression of SLC45A3 reduces myelin protein expression in mature oligodendrocytes and alters oligodendrocyte morphology, indicating that tight regulation of SLC45A3 expression is necessary for the proper maintenance of myelin proteins and structure. Taken together, our data suggest that miR-32 and its downstream target SLC45A3 play important roles in myelin maintenance by modulating glucose and lipid metabolism and myelin protein expression in oligodendrocytes.

Fig. 1

miR-32 promotes myelin protein expression in OLs and is dysregulated in the OL-specific Dicer KO. (A) Ultrastructure of adult spinal cord indicates that loss of Dicer results in thinner myelin and an absence of myelin in some axons. (B) Quantitative miRNA real time PCR. miR-32 expression is increased in mature OLs compared to premature OLs (OPCs), but miR-144 does not show a difference. (C) Developmental expression of miR-32. Total RNAs from postnatal mouse brains (1, 7, 21, 30, and 50 day-old) were used for the syntheses of cDNAs and then analyzed by individual miRNA-specific real time PCRs. Data were normalized by the result of U6 control. (D) In situ hybridization against mmu-miR-32 on mouse brain sagittal sections. Suppression of miR-32 reduces myelin protein expression (E) and ectopic overexpression of miR-32 increases myelin protein expression (F). Whole cell extracts were analyzed by Western blot with antibodies against myelin specific proteins. OPCs were differentiated into mature forms with tri-iodothyronine in vitro for 2 days, transduced with the lentivirus harboring miR-32 shRNA (E) or transcript (F), differentiated with tri-iodothyronine for 5 more days (total 7 days in vitro differentiation), and then stained with MBP antibody (red). miR-32 suppression downregulates OL maturation, and conversely its overexpression upregulates OL differentiation with more myelin membrane-like sheets in culture. DIV stands for ‘days in vitro differentiation’. Both GAPDH and GFP were used for the normalization controls.

Fig. 2

SLC45A3 is a myelin-enriched miR-32 target protein. (A) miR-32 represses cldn11 and slc45a3 expression in a dosage dependent manner through its 3’UTR binding site. (B) Knocking down miR-32 increases SLC45A3 levels. (C) SLC45A3 is increased in myelin (plus membrane) fraction of the OL-specific Dicer knockout mice. (D) SLC45A3 is highly upregulated in OL-specific Dicer ablated mouse brain. CC1 is an OL marker (green) and SLC45A3 is red.

Fig. 3

SLC45A3 regulates glucose and lipid metabolism. (A) shRNA or cDNA of slc45a3 was transduced with lentivirus into the OL cells followed by intracellular glucose level measurements. Serial increases of slc45a3 shRNA reduced glucose and conversely, overexpression upregulated glucose levels. (B) Exogenous overexpression of slc45a3 in adipocytes upregulated the amount of intracellular free fatty acids. Palmitic acid is highly increased by SLC45A3 overexpression in a dose dependent manner. (C) SLC45A3 increases neutral lipid accumulation in the adipocytes, which were tested by Oil Red O stain. (D) Quantities of accumulated lipid droplets were measured by optical densities (OD) of the extracted lipids at 492 nm. Total neutral lipids in the SLC45A3 overexpressed adipocytes were increased about 20% compared to controls.

Fig. 4

Modulated SLC45A3 expression is necessary for proper OL maintenance. OPCs were differentiated into mature forms with tri-iodothyronine in vitro for 2 days, transduced with the lentivirus harboring miR-32 shRNA (E) or transcript (F), differentiated with tri-iodothyronine for 5 more days (total 7 days in vitro differentiation), and then analyzed with myelin specific antibodies for western blot (A and C) and MBP antibody for immunocytochemistry (B and D). Overexpression of SLC45A3 reduces myelin protein levels (A) and mature forms of OLs (B). Suppression of SLC45A3 also results in abnormal myelin protein production (C) and less mature OLs in vitro (D), suggesting moderate SLC45A3 expression is necessary for proper OL development and/or function. (E) Model of the involvement of miR-32 and SLC45A3 in the regulation of myelin lipid metabolism. miR-32 may fine-tune SLC45A3 expression in OLs, which regulates proper myelin maintenance through OL differentiation and lipid metabolism/biogenesis. There might be also other proteins including CLDN11 regulated by miR-32 that affect the physiology of OLs and myelin. DIV stands for ‘days in vitro differentiation’.