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High cholesterol turnover catalyzed by cholesterol 24-hydroxylase is essential for neural functions, especially learning. Because 24(S)-hydroxycholesterol (24-OHC), produced by 24-hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH-SY5Y neuron-like cells as a model, we examined whether 24-OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24-OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24-OHC efflux was stimulated in the presence of high-density lipoprotein (HDL), whereas apolipoprotein A-I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24-OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A-I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24-OHC. These results suggest that ABCA1 actively eliminates 24-OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.
The brain contains ~ 25% of the human body's total cholesterol, despite occupying only 2% of body mass. Some fraction of cholesterol is actively converted to 24(S)-hydroxycholesterol (24-OHC) by cholesterol 24-hydroxylase, a cytochrome P450 (CYP46A1) highly expressed in a subset of neurons in the brain, and subsequently eliminated from brain tissue (Bjorkhem et al. 1997); about 0.02% of brain cholesterol in humans and 0.4% in mouse turns over each day (Dietschy and Turley 2004). Disruption of the mouse CYP46A1 gene reduced the synthesis of new cholesterol in the brain by ~ 40%, indicating at least 40% of cholesterol turnover in the brain is dependent on the conversion into 24-OHC (Lund et al. 2003). This knockout mouse exhibited severe deficiencies in spatial, associative, and motor learning, as well as in hippocampal long-term potentiation (Kotti et al. 2006), suggesting that cholesterol turnover via 24-hydroxylase is essential for brain functions, especially learning.
Because hydroxylation of the side chain of cholesterol accelerates transfer across the lipid bilayer more than 1000 times relative to unhydroxylated cholesterol (Meaney et al. 2002), it is generally accepted that after its production in the brain, 24-OHC gains access to the circulation by spontaneous diffusion across cellular membranes and the blood–brain barrier (Russell et al. 2009); however, some studies have demonstrated transporter-mediated oxysterol efflux (Tam et al. 2006; Ohtsuki et al. 2007; Terasaka et al. 2007). Tam et al. reported that ABCA1 mediates the efflux of 25-hydroxycholesterol from human embryonic kidney (HEK) cells expressing ABCA1 as well as from mouse primary embryonic fibroblasts (Tam et al. 2006); Terasaka et al. reported that ABCG1 promotes the efflux of 7-ketocholesterol and other oxysterols from macrophages onto high-density lipoprotein (HDL), and protects these cells against apoptosis induced by oxidized low-density lipoprotein (Terasaka et al. 2007).
Although most of hydrophobic and amphipathic compounds pass freely through the lipid bilayer, some ABC proteins, such as ABCB1 (MDR1) and ABCG2, actively transport hydrophobic and amphipathic compounds, thereby playing important roles in protecting our body by expelling such compounds into the intestinal lumen, into the bile from the liver, and into the urine from the kidney (Ueda 2011). ABCA1 and ABCG1 are involved in the efflux of cholesterol from cells. ABCA1 mediates the efflux of cholesterol to lipid-free apolipoprotein A-I (apoA-I), which serves as a lipid acceptor in the serum (Wang et al. 2000; Tanaka et al. 2001); in the case of ABCG1, HDL acts as the acceptor (Wang et al. 2004; Vaughan and Oram 2005; Kobayashi et al. 2006). Both ABCA1 and ABCG1 are expressed in the brain (Fukumoto et al. 2002; Koldamova et al. 2003; Tachikawa et al. 2005; Tarr and Edwards 2008). ApoE-containing lipoproteins, HDL-like particles, function in delivery of cholesterol from astrocytes to neurons in brain, while HDL containing apoA-I functions in reverse cholesterol transport in peripheral tissues. ABCA1 and ABCG1 expressed in astrocytes are involved in the formation of apoE-containing lipoproteins (Hirsch-Reinshagen et al. 2004; Karten et al. 2006), which stimulate axonal extension of neurons (Matsuo et al. 2012). The importance of ABCA1 and ABCG1 for the formation of apoE-containing lipoproteins is shown in results that apoE and cholesterol levels decrease in cerebrospinal fluid (CSF) of Abca1 knockout mice (Wahrle et al. 2004) and cholesterol level in astrocyte increases in Abcg1 knockout mice (Wang et al. 2008). Both proteins are also expressed in neurons, where their physiological roles remain unclear. We examined the possible roles of ABCA1 and ABCG1 in 24-OHC efflux from neuronal cells, using SH-SY5Y cells as a model. We found that both ABC proteins play roles in 24-OHC efflux.
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- Materials and methods
- Supporting Information
24-OHC is the major cholesterol metabolite in the brain (Bjorkhem et al. 1997); high cholesterol turnover catalyzed by cholesterol 24-hydroxylase is essential for neural functions, especially learning (Kotti et al. 2006). We hypothesized that 24-OHC produced in neuronal cells is actively eliminated via transporters; in this study, we found that ABCA1 transports 24-OHC from differentiated SH-SY5Y neuron-like cells in an HDL-dependent manner.
We predicted that 24-OHC would be eliminated by the transporter(s) whose expression is induced by intracellular accumulation of 24-OHC itself. 24-OHC is an endogenous ligand of a nuclear receptor LXR; 24-OHC therefore induces transcription of ABCA1 and ABCG1 genes in macrophages and neuronal cells by activating the LXR/RXR heterodimer (Repa et al. 2000; Bjorkhem and Meaney 2004). However, it was reported that 24-OHC is not produced in SH-SY5Y cells (Ohyama et al. 2006), and indeed we could not detect 24-OHC production under our experimental conditions (data not shown). Therefore, we added 24-OHC exogenously and examined if 24-OHC induces expression of ABCA1 and ABCG1. ABCA1 and ABCG1 genes in differentiated SH-SY5Y cells well responded to exogenously added 24-OHC and synthetic ligands and 24-OHC (10 μM) induced them as efficiently as the synthetic ligands (Fig. 1). This concentration is physiologically relevant, as free 24-OHC in the brain was estimated at up to 30 μM (Lutjohann et al. 1996; Kolsch et al. 1999).
24-OHC efflux was clearly observed in the presence of HDL even without TO + RA treatment, and this efflux was further stimulated by the treatment (Fig. 2c). 24-OHC efflux in the presence of apoA-I was quite low compared to efflux in the presence of HDL. Because cholesterol efflux by ABCA1 is dependent on lipid-free apolipoproteins, and because efflux by ABCG1 is HDL-dependent, it was possible that ABCG1 is responsible for 24-OHC efflux from differentiated SH-SY5Y cells.
To examine if 24-OHC efflux to HDL was mediated by ABCG1, we suppressed the expression of ABCG1 and ABCA1 using siRNAs. Surprisingly, siRNA against ABCG1 had no significant effect, whereas siRNA against ABCA1 decreased 24-OHC efflux from differentiated SH-SY5Y cells in the presence of HDL. Because ABCA1 expression was induced when ABCG1 expression was suppressed, we speculate that increased levels of ABCA1 compensated for the lower levels of ABCG1 (Fig. 4a). However, when ABCA1 expression was suppressed, the 24-OHC efflux did not correlate with ABCG1 expression. Therefore, the involvement of ABCG1 in 24-OHC efflux from differentiated SH-SY5Y cells remains unclear. To determine the involvement of ABCA1 and ABCG1 in 24-OHC efflux, we studied HEK293 cells stably expressing ABCA1 or ABCG1 (Figs 5 and 6). 24-OHC efflux from HEK/ABCA1 and HEK/ABCG1 was clearly observed in the presence of HDL, whereas efflux in the presence of apoA-I was marginal. Taken together, these results suggest that both ABCA1 and ABCG1 mediate 24-OHC efflux to HDL.
Because cholesterol efflux by ABCA1 is apoA-I–dependent (Wang et al. 2000; Tanaka et al. 2001), it was surprising to observe 24-OHC efflux by ABCA1 in the presence of HDL, but not apoA-I. However, we previously reported (Nagao et al. 2009) that the function of ABCA1 does not depend on apoA-I, and that ABCA1 can transport cholesterol in the presence of bile salts in the medium. We have also proposed that lipid accumulation within the extracellular domain via ATP hydrolysis-dependent lipid transport causes conformational changes that generate apoA-I–binding site(s) on the surface of the extracellular domain of ABCA1, and that apoA-I bound to these site(s) is directly loaded with lipids by ABCA1 (Nagao et al. 2012). The amphipathic molecule 24-OHC may not be reserved in the extracellular domain of ABCA1, but may escape to the medium and bind to HDL as a lipid acceptor in the medium.
Because 24-OHC induces apoptosis of neuronal cells by generating free radicals (Kolsch et al. 2001), 24-OHC must be eliminated from cells as soon as possible after its production by 24-hydroxylase. Our study is the first to demonstrate that 24-OHC is actively eliminated by ABCA1. Treatment with ABCA1 siRNAs stimulated ABCG1 expression; simultaneous treatment with siRNAs against ABCA1 and ABCG1 negatively affected the survival of differentiated SH-SY5Y cells, especially in the presence of 24-OHC. We observed 24-OHC efflux from HEK/ABCG1 cells in the presence of HDL; therefore, ABCG1 may also be involved in 24-OHC elimination. The treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24-OHC, suggesting that expression of ABCA1 and ABCG1 prevent apoptosis of neuronal cells. This should be also examined using neuronal cells from the ABCA1/ABCG1 double knockout mouse. ABCG4 was not analyzed in this study, since no antibody, which recognizes endogenously expressed ABCG4, was available. Because ABCG4 is expressed in brain and is required for sterol transport (Wang et al. 2008), it is possible that ABCG4 is also involved in 24-OHC efflux.
The G-395C polymorphism in the promoter of ABCA1 is implicated in low serum HDL levels (Probst et al. 2004) and reduces the CSF concentration of 24-OHC (Kolsch et al. 2006). This reduction may be caused by decreased 24-OHC efflux by ABCA1, or by lower levels of HDL, which serves as a lipid acceptor in the CSF. Specific inactivation of mouse brain ABCA1 leads to motor and sensorimotor behavioral and synaptic changes (Karasinska et al. 2009). The results reported in this study will enhance our understanding of the roles of ABC proteins and HDL in cholesterol homeostasis in the brain.