Cholesterol metabolism in neurons and astrocytes

Progress in Lipid Research 50 (2011) 357–371

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Progress in Lipid Research journal homepage: www.elsevier.com/locate/plipres

Review

Cholesterol metabolism in neurons and astrocytes Frank W. Pfrieger ⇑, Nicole Ungerer CNRS UPR 3212, University of Strasbourg, Institute of Cellular and Integrative Neurosciences (INCI), 67084 Strasbourg Cedex, France

a r t i c l e

i n f o

Article history: Received 17 March 2011 Received in revised form 11 June 2011 Accepted 22 June 2011 Available online 1 July 2011 Keywords: Neuroglia Membrane lipids Steroids Apolipoproteins Lipoproteins Neurodegenerative diseases

a b s t r a c t Cells in the mammalian body must accurately maintain their content of cholesterol, which is an essential membrane component and precursor for vital signalling molecules. Outside the brain, cholesterol homeostasis is guaranteed by a lipoprotein shuttle between the liver, intestine and other organs via the blood circulation. Cells inside the brain are cut off from this circuit by the blood–brain barrier and must regulate their cholesterol content in a different manner. Here, we review how this is accomplished by neurons and astrocytes, two cell types of the central nervous system, whose cooperation is essential for normal brain development and function. The key observation is a remarkable cell-specific distribution of proteins that mediate different steps of cholesterol metabolism. This form of metabolic compartmentalization identifies astrocytes as net producers of cholesterol and neurons as consumers with unique means to prevent cholesterol overload. The idea that cholesterol turnover in neurons depends on close cooperation with astrocytes raises new questions that need to be addressed by new experimental approaches to monitor and manipulate cholesterol homeostasis in a cell-specific manner. We conclude that an understanding of cholesterol metabolism in the brain and its role in disease requires a close look at individual cell types. Ó 2011 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurons and astrocytes: some background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-autonomous supply of cholesterol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cholesterol synthesis in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Cholesterol synthesis in neurons and astrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . External supply of cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Cholesterol secretion via lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Expression of apolipoproteins in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Lipoprotein secretion by astrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Properties of astrocytic lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. Mechanisms of lipoprotein secretion by astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Receptor-mediated uptake of lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Expression of lipoprotein receptors in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Receptor-mediated lipoprotein uptake by neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Intracellular redistribution of lipoprotein-derived cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Handling surplus cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Storage after esterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Elimination of cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: 24OHC, 24S-hydroxycholesterol; ABC, ATP binding cassette; CSF, cerebrospinal fluid; CNS, central nervous system; DRG, dorsal root ganglion; ER, endoplasmic reticulum; LXR, liver x receptor; RGCs, retinal ganglion cells; VLDL, very low density lipoproteins. ⇑ Corresponding author. Address: CNRS UPR 3212, University of Strasbourg, Institute of Cellular and Integrative Neurosciences (INCI), 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France. Tel.: +33 388456645; fax: +33 388601664. E-mail addresses: [email protected], [email protected] (F.W. Pfrieger). 0163-7827/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2011.06.002

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5.2.1. Conversion to an oxysterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Secretion via ABC transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Cholesterol is an indispensible component of biological membranes and precursor to numerous signalling molecules including steroid hormones. Its provision and disposal in all organs of the mammalian body – except for the brain – relies on dietary uptake by the intestine, on de novo synthesis in every organ, and on lipoprotein-mediated transport via the blood circulation. Cells in the brain are cut off from this elaborate system by the blood–brain barrier, which prevents lipoprotein exchange [1,2]. Therefore, cells in the brain have implemented their specific way to handle cholesterol turnover. Here, we summarize current knowledge of cholesterol metabolism in the central nervous system (CNS) with a focus on two cell types, namely neurons and astrocytes (Fig. 1). Their cooperation is essential for normal brain function, and a disturbance of their interactions can provoke pathologic changes. Complementary overviews focus on cholesterol metabolism in the nervous system [3–8] and possible links to brain injury and diseases [4,9–18].

2. Neurons and astrocytes: some background The brain consists of neurons and glial cells. Neurons specialize in the generation and transmission of electrical signals that represent the basis of all brain functions. To accomplish this, they form elaborate processes called axons and dendrites and complex intercellular connections called synapses (Fig. 2). Glial cells, which comprise astrocytes, oligodendrocytes, ependymal and microglial cells,

Fig. 1. Neurons and astrocytes. The drawing by the Spanish neuroscientist and Nobel prize winner Santiago Ramón y Cajal illustrates human hippocampal protoplasmic astrocytes (A and B) that embrace cell bodies and dendrites of pyramidal neurons (C and D). The drawing was made after light microscopic study of an autopsy sample subjected to histologic gold chloride-sublimate staining. Reprinted with permission from [336].

Fig. 2. Spatial arrangement of astrocytic processes and chemical synapses. The electron micrograph of the molecular layer of the monkey cerebellum shows astrocytic processes (red) and synaptic connections between a presynaptic neuron that forms vesicle-filled terminals (blue) and a postsynaptic neuron with receptorbearing spines (green). Scale bar, 200 nm. Reprinted with permission from [337].

provide structural and logistic support to neurons, which allows them to develop and function properly [19,20]. Among the glial cells, we focus on astrocytes, which form a complex fabric that enwraps neurons (Fig. 2) and ensure that neurons can generate and transmit electrical signals [21]. To this end, astrocytes regulate the extracellular potassium concentration [22], maintain the neuronal transmitter pool of glutamate by the glutamine–glutamate cycle [23], provide neurons with energy substrates and antioxidative substances [24,25] and mediate the activity-dependent regulation of cerebral blood flow [26,27]. Moreover, astrocytes promote the formation of synapses [28] and sense or even influence their activity [29–34]. Last but not least, there is evidence that astrocytes play a role in neurodegeneration [21,35–38]. Neurons and astrocytes have a very high demand for cholesterol. Neurons need to build the enormous membrane surface of their axons, dendrites and synapses [39,40]. For example, the membrane area of cerebellar Purkinje cells, namely their dendrites, reaches up to 150,000 lm2 [41], whereas myocyte membranes comprise only 5000 lm2 [42]. Synapses contain large amounts of membrane in postsynaptic spines and presynaptic vesicles, which have a particularly high cholesterol content (40 mol%) [43]. Individual astrocytes require large amounts of membrane, as they occupy immense non-overlapping volumes and touch up to 100,000 synapses through fine, micrometer sized processes [44–48]. The demand of neurons and astrocytes for cholesterol in the adult brain cannot be measured directly. Previous studies indicated that the half-life of cholesterol in the brain lasts between two to six months [49,50], which indicated that there is little turnover of cholesterol in the brain. However, the bulk of cholesterol in the brain is contained in myelin, and therefore estimates of the metabolic stability of cholesterol in whole brain samples are largely determined by this pool [2]. The turnover of cholesterol in individual neurons and astrocytes may in fact be very high and reach an estimated 20% per day depending on the brain area and the neuronal cell type [2]. Support for this conclusion comes from the fact that the turnover of cholesterol is proportional to the metabolic rate across different tissues and animal species [2]. Since neurons have an intense

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metabolic activity to generate action potentials and synaptic signals [51–53], their cholesterol turnover should be high as well. 3. Cell-autonomous supply of cholesterol All cholesterol contained in the CNS must be formed in situ, because brain cells have no access to the hepatic or dietary supply of cholesterol due to the blood–brain barrier. In general, mammalian cells can synthesize cholesterol from acetyl coenzyme A by a complex series of reactions that are catalyzed by more than 20 enzymes and that require energy and molecular oxygen [54]. In the following, we will describe, whether and how neurons and astrocytes use de novo synthesis to meet their need for cholesterol. 3.1. Cholesterol synthesis in the brain First evidence that brain cells synthesize cholesterol has been published 60 years ago by the group of Heinrich Waelsch. Their pioneering use of heavy water to study lipid synthesis in rats indicated that cholesterol is synthesized in the brain and that the rate of synthesis is higher during early postnatal development than in adult animals [55,56]. Using similar methods and thorough quantification, Quan and colleagues revealed that cholesterol synthesis in the developing mouse brain peaks during the second postnatal week and varies across regions. In adults, it falls to a tenth of the peak value and remains unaffected by gender or the cholesterol concentration in the blood [57]. Post-mortem examination of human brains reported an age-related decline of specific cholesterol precursors and concluded that cholesterol synthesis decreases with age [58]. A pioneering study indicated that the extent of cholesterol synthesis changes after brain injury [59]. Following a lesion of the entorhinal cortex of adult rats, the activity of 3hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), which catalyzes the rate-limiting step in cholesterol synthesis, declined within eight days and returned to a normal level within a month. At the same time, proteins involved in cholesterol release and uptake were upregulated, which suggested that post-lesion recycling rather than synthesis of cholesterol is required for the regeneration of synaptic connections [59]. The importance of cholesterol synthesis for brain function is underlined by the fact that genetic defects in enzymes that mediate the post-squalene biosynthesis of cholesterol cause neurologic symptoms [60]. The most frequent and best studied disease is the Smith–Lemli–Opitz syndrome, an autosomal recessive disorder that is characterized by malformation of several organs, neurologic symptoms and behavioral abnormalities. The genetic defect concerns 7-dehydrocholesterol reductase, which catalyzes a last step in cholesterol biosynthesis [61] (Fig. 3). 3.2. Cholesterol synthesis in neurons and astrocytes The extent of cholesterol synthesis in neurons and astrocytes in vivo remains unknown, because existing methods like metabolic labeling or pharmacologic manipulations do not allow to study cholesterol formation in specific cell types. Within the last 5 years, a new approach has appeared on the scene, which is based on the cell type-specific ablation of squalene synthase (farnesyl diphosphate farnesyl transferase 1, SQS/FDFT1) using the Cre/loxP system [62–64]. SQS/FDFT1 catalyzes the first committed step in sterol biosynthesis [65]. Saito and colleagues used this approach to block cholesterol biosynthesis in neuronal precursor cells during embryonic development by a Cre recombinase expressing mouse line, which targets progenitor cells in the ventricular zone and their progeny using the Nestin promoter. Mutant mice showed reduced brain size and perinatal lethality and newly generated neurons, but not their precursors, died of apoptosis. This indicated that new-

Fig. 3. Cholesterol synthesis in neurons and astrocytes. The diagram illustrates distinct features of cholesterol synthesis in neurons (blue) and astrocytes (red). Selected enzymatic steps and precursors in the biosynthesis of cholesterol that show cell-specific distribution based on in vitro data. The two pathways of cholesterol synthesis differ in the sequence of enzymatic steps and the resulting precursors. Enzymes: 1, SQS/FDFT1, squalene synthase (EC 2.5.1.21); 2, DHCR24, 24-dehydrocholesterol reductase (EC 1.3.1.72); 3, CYP51, cytochrome P450, family 51 (lanosterol 14-demethylase; EC 1.14.13.70); 4, NSDHL, NAD(P) dependent steroid dehydrogenase-like (EC 1.1.1.170, part of C4-demethylation enzyme complex). Precursors: 7D, 7-dehydro-cholesterol (KEGG entry C01164); CH, cholesterol (C00187); DE, desmosterol (C01802); LA, lanosterol (C01724); LT, lathosterol (C01189); ZN, zymostenol (C03845); ZY, zymosterol (C05437).

born neurons must synthesize cholesterol cell-autonomously in order to survive [64]. Fünfschilling and colleagues used a similar approach to ablate Sqs/Fdft1 in the adult brain using the Tg(Malpha6-Cre) line [63]. The transgenic mice targeted virtually all postmitotic, postmigratory cerebellar granule cells as well as neurons in brainstem nuclei. Absence of SQS/FDFT1 from these neurons had no effect on cerebellar morphology or function, and the mice showed normal motor control. Notably, absence of Sqs/Fdft1 expression in granule cells did not change the protein content of SQS/FDFT1 in cerebellar lysates indicating that these cells do not produce the enzyme at the adult stage. Sterol synthesis in neurons and astrocytes in vivo may be estimated from transcript profiles of cholesterol synthesizing enzymes. A recent analysis of in situ hybridization data from the Allen Mouse Brain Atlas (http://www.brain-map.org) suggests that transcript levels of many cholesterol biosynthetic enzymes are higher in neurons compared to astrocytes [66]. However, these data should be interpreted with caution, as correlations between mRNA and protein abundance [67] and between protein levels and enzymatic activity are unknown. Cell culture experiments can address aspects of cholesterol biosynthesis in neurons and astrocytes that cannot be studied in vivo, although we note that culture conditions can influence cholesterol homeostasis. Several groups compared sterol synthesis in neuronal and glial cell preparations using metabolic labeling with radioactive precursors. Their studies reported marked, albeit conflicting, differences in terms of efficacy and regulation [68–73]. Nieweg and colleagues used serum-free cultures of highly purified CNS neurons and glial cells from postnatal rats. They detected different profiles of post-squalene precursors in neurons compared to glial cells (Fig. 3) [73]. Neurons contained mainly sterols of the socalled Kandutsch–Russel pathway, whereas astrocytes contained precursors of the Bloch

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pathway (Fig. 3). The accumulation of desmosterol in cultured astrocytes has been described previously [72,74,75] suggesting that desmosterol allows to trace sterol synthesis by this glial cell type. The high desmosterol content of the brain during postnatal development [57,76] may reflect sterol formation by newborn astrocytes during this period. Notably, Nieweg and colleagues observed a lower rate of sterol synthesis in neurons compared to glial cells. In neurons, radioactive label was mainly found in lanosterol, whereas in glial cells it accumulated predominantly in cholesterol (Fig. 3) [73]. The accumulation of lanosterol in neurons is in line with previous reports [72,77]. Nieweg and colleagues detected very low levels of lanosterol-converting enzymes, 24-dehydrocholesterol reductase (seladin-1, DHCR24) and lanosterol 14-alpha demethylase (cytochrome P450, family 51, CYP51) suggesting that neurons cannot convert lanosterol efficiently (Fig. 3) [73]. Moreover, inhibition of FDFT1 induced a much weaker up-regulation of biosynthetic enzymes in neurons compared to astrocytes. This suggests that neurons have a lower capacity to compensate for a cholesterol deficit by de novo synthesis compared to astrocytes. Another question that cannot be addressed in vivo is whether axons and dendrites synthesize cholesterol autonomously. This was studied in so-called compartmented cultures, where neuronal axons and somata can be analyzed separately. Using such cultures of sympathetic neurons from newborn rats, Vance and colleagues showed that cholesterol synthesis is restricted to neuronal somata and does not occur in axons, whereas formation of phospholipids occurred in both compartments [78]. The distribution and activity of cholesterol-synthesizing enzymes in axons and dendrites still needs to be elucidated. Both compartments contain endoplasmic reticulum (ER) [79], where cholesterol synthesizing enzymes are located. Previous studies indicated a rapid intracellular redistribution of newly synthesized cholesterol in neurons and astrocytes. Metabolic labeling of cultured rat retinal ganglion cells (RGCs) and astrocytes revealed that newly synthesized cholesterol and precursors are transferred to the plasma membrane [73]. A study on compartmented cultures of rat sympathetic neurons revealed that newly synthesized cholesterol is transported from the soma to the axon [80]. In general, the subcellular dispatch of newly synthesized cholesterol from the ER to the plasma membrane is mediated by non-vesicular mechanisms that may involve direct membrane contact or specialized cytosolic carriers [81–84]. At present, it remains to be studied whether and how neurons and astrocytes implement this transport system. Their complex morphology may demand elaborate mechanisms for the distribution of newly synthesized cholesterol into their vast cellular space. In summary, studies on cell-specific knockout-mice indicate that during embryonic development, newborn neurons must produce cholesterol in a cell-autonomous manner to survive, whereas in the adult brain, they acquire cholesterol from other sources. In vitro experiments suggest that neurons and astrocytes differ in the precursor profile of cholesterol synthesis and that astrocytes produce cholesterol at higher rates (Fig. 3). 4. External supply of cholesterol As alternative to de novo synthesis, mammalian cells can import cholesterol from an external source. To this end, providing cells secrete cholesterol as lipoproteins, which are taken up by target cells via specific receptors [83]. The following paragraphs summarize, whether and how neurons and astrocytes use this form of cholesterol provision. 4.1. Cholesterol secretion via lipoproteins If cells in the brain rely on the import of cholesterol, there must be a cell type that secretes cholesterol-containing lipoproteins.

There is strong experimental evidence that astrocytes fulfill this role in the CNS. 4.1.1. Expression of apolipoproteins in the brain The expression of apolipoproteins should identify cells that secrete lipids via lipoproteins. The mammalian brain contains a subset of the apolipoproteins that are present in the blood. This includes apolipoprotein E (APOE) [85,86], apolipoprotein J/clusterin (APOJ/ CLU) [87,88] and apolipoprotein D (APOD) [89]. Apolipoprotein A1 (APOA1) is synthesized by endothelial cells of brain capillaries and by cells of the choroid plexus epithelium, but not by astrocytes or neurons [85,90–92]. It is present in cerebrospinal fluid [93,94], but barely detectable in lysates of rodent brain [95,96]. There is solid evidence that under normal conditions, APOE is produced exclusively by astrocytes and by astroglial cells like cerebellar Bergmann glia, tanycytes and retinal Müller cells [95,97–99]. Moreover, astrocytes express APOJ/CLU and APOD [100], which have also been detected in subsets of neurons in rodents and humans [89,101–111]. At present, it is unclear, whether APOD binds cholesterol. Its expression is highly upregulated in mice lacking APOE suggesting its involvement in lipoprotein metabolism [112]. Previous studies have shown that various forms of injury and disease cause upregulation of APOE, APOD and APOJ/CLU in the mammalian CNS [103,106,110,113–120]. The increase in APOE occurred mainly in glial fibrillary acidic protein (GFAP)-positive cells [113,121–126], but it was also observed in neurons [127]. The appearance of APOE in neurons after injury or disease could be due to increased uptake rather than enhanced expression [128,129]. Direct evidence for lesion-induced neuronal expression of APOE comes from a study on a transgenic knock-in mouse, where the expression of green fluorescent protein is controlled by the Apoe promoter. Administration of kainic acid induced APOE expression in hippocampal neurons [98]. Interestingly, APOE expression was decreased in primary astrocytes derived from a mouse model of Huntington’s disease [130]. An injury- or disease-induced increase in APOD was observed in astrocytes and neurons [106,109,131–133]. Arraybased transcript profiling and behavioral testing in rats revealed an age-dependent impairment of performance in a spatial memory task that was accompanied by increasing levels of APOE in hippocampal astrocytes [134]. Possible correlations between cholesterol transport and age-related cognitive decline need to be further explored. 4.1.2. Lipoprotein secretion by astrocytes There is good evidence that astrocytes secrete lipoproteins in vivo. In a pioneering study, Amaratunga and colleagues showed that retinal Müller cells, which represent a subtype of astroglial cells, produce and secrete APOE in vitro, and that injection of radiolabeled amino acids in the retinal vitreous led to accumulation of labeled APOE in the optic nerve [135]. These results suggested that APOE secreted by retinal Müller cells in vivo is taken up by RGCs, whose axons form the optic nerve. Other studies detected lipoproteins that contain APOE in cerebrospinal fluid (CSF) of rodents and humans [8,93,94,136–140]. In humans, the APOE level in CSF is less than 5% of its concentration in plasma [93,94,140]. Since blood-derived lipoproteins cannot enter the brain and since APOE is made exclusively by astrocytes, these cells are most likely the source of the particles. Further support comes from a study on transgenic mice, which express human isoforms of APOE under the control of the human GFAP promoter. This promoter drives expression in subsets of astrocytes and neuronal precursors [141,142]. The CSF of these mice contained lipoproteins with human isoforms of APOE indicating their provenance from astrocytes [139]. In cultured astrocytes, APOE deficiency abolished lipoprotein secretion [143,144], but its impact on lipoprotein secretion in vivo remains to be clarified. Studies on APOE-deficient mice [145,146] revealed

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normal cholesterol content [112,147,148] and turnover in various brain regions [57]. On the other hand, mice lacking APOE showed significantly reduced levels of cholesterol and selected precursors including desmosterol in the hippocampus compared to control mice, when maintained in a normal environment, but enhanced levels when maintained in an enriched environment [149]. This indicated an influence of APOE on cholesterol metabolism and its modification by sensory stimulation. Interestingly, APOE-deficient mice show behavioral and neurologic symptoms [150–158] and defects in sensory systems [159–163]. At the cellular level, APOE deficiency causes an agedependent loss of synapses and dendrites [164,165] and a shift in the transbilayer distribution of cholesterol at synaptic membranes [166]. It remains unknown, whether these changes are caused by reduced lipoprotein secretion from astrocytes or by defects in APOE signaling that are independent from cholesterol. APOJ/CLU-deficient mice show no overt changes in the nervous system [167], but reduced damage due to hypoxia–ischemia-induced brain injury suggesting that this protein contributes to neuronal cell death [168]. 4.1.3. Properties of astrocytic lipoproteins Studies of astrocyte-derived lipoproteins formed in vitro revealed that their chemical and physical properties differ substantially from those present in plasma [74,94,138,139,143,169–173]. Lipoproteins secreted by cultured astrocytes contain cholesterol and phospholipids, but comparatively little esterified cholesterol or triglycerides. The presence of cholesterol precursors, namely lathosterol and desmosterol, in glia-derived lipoproteins [74,75] suggests that astrocytes secrete precursors that are taken up by neurons and converted to cholesterol. Such partial outsourcing may be very efficient, as it circumvents inefficient steps in neurons like the conversion of lanosterol. Astrocyte-derived lipoproteins are disc-shaped with sizes ranging from 8 to 17 nm, and their densities are similar to those of high-density lipoproteins [138]. They contain only APOE and APOJ/CLU, which are present on distinct particles [104,138,139,143,169]. APOE particles are larger and contain more lipid than those harboring APOJ/CLU [139,143]. Cultures prepared from knockout mice showed that expression of APOE, but not of APOJ/CLU is required for lipid secretion [139,143,144]. So far, only one study showed that cultured astrocytes secrete APOD associated with lipids [105] and therefore, APOD-containing complexes should be characterized further. The properties of lipoproteins that are produced by astrocytes in vivo are currently unknown due to the lack of suitable isolation methods. They may differ from those produced in vitro for two reasons. First, cultured astrocytes are mainly surrounded by medium, whereas astrocytes in vivo are embedded in a narrow and tortuous extracellular space, which may influence the size and composition of their lipoproteins. Second, cultured astrocytes are generated from precursors in vitro in the presence of serum, and may not fully recapitulate the differentiation of astrocytes in vivo as indicated by a recent transcript analysis [174]. The properties of astrocytic lipoproteins produced in vivo may also differ from those present in CSF. The CSF harbors a variety of lipoproteins with distinct physical and chemical properties, which may be modified by the isolation methods [94,138–140]. A subclass of CSF lipoproteins loosely resembles those secreted by astrocytes in vitro [138,139] suggesting their astrocytic origin [175]. Astrocyte-derived particles may undergo modifications in the parenchym or in the CSF [176]. This includes esterification of cholesterol by secreted lecithin cholesterol acyltransferase (LCAT), which allows the molecule to enter the hydrophobic core of lipoproteins. This enzyme was found to be expressed by neurons and glial cells in adult rhesus monkeys [101] and by cultured neurons and astrocytes from embryonic and postnatal mice [177]. LCAT activity was detected in human CSF [178–180], and its absence

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in knockout mice lowered the concentration of cholesteryl esters in the brain compared to wildtype controls [181]. A recent in vitro study showed that despite its expression in neurons and astrocytes, LCAT was only present in astrocyte-conditioned medium [182]. The enzyme esterified preferentially cholesterol in culture medium from astrocytes rather than from neurons and APOE activated the enzyme. Finally, lipoprotein particles from glial cells of LCAT-deficient mice were smaller than those secreted by glial cells from wildtype mice [182]. In agreement with these data, cholesterol esterification was detected only in astrocyte-, but not in neuron-conditioned culture medium [73].

4.1.4. Mechanisms of lipoprotein secretion by astrocytes It is unclear, how lipoproteins are assembled and secreted by astrocytes. The mechanisms may be similar to those employed by hepatocytes to secrete lipoproteins, i.e. assembly of a core particle in the ER, transfer to the Golgi apparatus for further modifications and subsequent secretion [183]. These nascent lipoproteins may be directly taken up by neighboring neurons or they may acquire additional cholesterol by similar mechanisms as high-density lipoproteins [184]. Lipidation of nascent particles is mediated by specific subtypes of the ATP binding cassette (ABC) transporters. ABCA1 transfers cellular cholesterol to acceptors like APOA1 [185], and ABCG1 is thought to charge high-density lipoproteins with lipids [83,186–188]. Immunohistochemical staining showed that in adult rodents ABCA1 is predominantly expressed by neurons and at lower levels by glial cells [189–191]. The same appears to be true for ABCG1 and ABCG4 as shown by analysis of knock-in mice, where betagalactosidase replaces endogenous ABC transporters [192,193]. Cultured astrocytes express all three transporters at the transcript and protein level [75,144,191,192,194,195]. ABCG1 and ABCG4 colocalize in endosomal vesicles of cultured astrocytes, but are absent from the Golgi apparatus, the ER and from lysosomes [192]. Therefore, the pool of cholesterol that is accessible to these transporters, remains to be identified. Several studies have shown that efflux of cholesterol from cultured astrocytes can be enhanced by treatment with agonists of liver x receptors (LXRs) [144,191,194,196]. These nuclear receptors control expression of proteins that mediate cellular cholesterol release including APOE, ABCA1 and APOA1 [197,198]. The contribution of ABCA1 to cholesterol efflux from cultured astrocytes was indicated by the increased expression after treatment with a LXR agonist [191,194,196,199]. Studies on transgenic mice lacking or over-expressing ABCA1 support the idea that the transporter contributes to the lipidation of APOE-containing lipoproteins, although its role in astrocytes remains to be clarified. ABCA1 deficiency in mice reduced the cholesterol content and size of APOE-containing CSF lipoproteins [200] and the cholesterol concentration in culture medium of astrocytes [200,201]. Conversely, overexpression of ABCA1 in the mouse brain increased the size of CSF lipoproteins and enhanced the cholesterol to APOE ratio in lipoproteins from cultured astrocytes [202]. ABCA1 deficiency also caused a strong reduction of APOE in the brain and CSF [96,200,201]. This may signify enhanced catabolism of APOE due to insufficient lipidation. The extent, to which ABCA1 deficiency decreased APOE levels, was highest in the hippocampus and lowest in the cerebellum [201] suggesting that the contribution of ABCA1 to APOE lipidation varies across brain regions. A genetic study on humans showed that a single nucleotide polymorphism in the Abca1 gene correlates highly with the level of cholesterol in the CSF [203]. Finally, absence of APOE enhanced the transcript level of Abca1 in the brain indicating that ABC transporters can compensate for the loss of APOE-mediated cholesterol release [112].

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The residual efflux of cholesterol from astrocytes lacking ABCA1 may be mediated by ABCG transporters. Astrocytes from mice lacking both ABCG1 and ABCG4 showed reduced release of cholesterol and desmosterol onto high-density lipoprotein particles [75] and in cultured cerebellar glia, the extent of cholesterol efflux correlated with the expression level of ABCG1, but not of ABCA1 [144]. On the other hand, overexpression of ABCG1 in the CNS did not affect the APOE content in the brain or the cholesterol efflux from cultured glial cells [204]. Interestingly, increasing the levels of ABCG1 in glial cells enhanced the ability of glia-derived lipoproteins to stimulate axonal growth in vitro [205]. Overexpression and reduction of ABCA1 or ABCG transporters decrease and enhance the levels of cholesterol precursors in the brain [75,96,193,204] suggesting a direct or indirect influence of ABC transporters on cholesterol synthesis or precursor efflux. At present, it is unclear, whether astrocytes secrete cholesterol at a constant level or whether the release is modified by external or internal factors. Previous studies revealed various molecules that modulate the release of APOE and cholesterol from cultured astrocytes. This includes steroids [206], cytokines [207], growth factors [208,209] and the cAMP-, protein kinase C- and phosphoinositide3-kinase/extracellular signal-regulated kinase 1/2-dependent pathways [210,211]. 4.2. Receptor-mediated uptake of lipoproteins The cellular uptake of lipoproteins is mediated by specific receptors. They form an ancient family of structurally related membrane-spanning proteins that bind APOE and other ligands [212] and that fulfill transport and signaling functions [213]. Among the different family members, two receptors are most likely involved in cholesterol transport in the brain, the prototypic lowdensity lipoprotein receptor (LDLR) and the low-density lipoprotein receptor-related protein 1 (LRP1).

4.2.2. Receptor-mediated lipoprotein uptake by neurons Evidence that neurons take up lipoproteins via dedicated receptors comes from in vitro and in vivo studies. Pioneering work showed that monoclonal antibodies against LDLR block the uptake of fluorescently labeled lipoproteins from regenerating sciatic nerves by neurons from dorsal root ganglia of embryonic rats [233] and the enhancement of neurite outgrowth by very low density lipoproteins (VLDL) in cultured rabbit DRG cells. The latter effect required the presence of lipids, as APOE alone showed no effect [222,234]. Studies on purified rat RGCs showed that low density lipoprotein receptor-related protein associated protein 1 (LRPAP1), an antagonist of lipoprotein receptors [235], inhibits the effects of glia-conditioned medium on synapse number [236] and axonal growth [228]. Similarly, LRPAP1 and antibodies to LRP1 inhibited the growth of neurites and dendrites in cultured neurons induced by a feeding layer of cortical astrocytes [230,237] or by blood-, CSFor glia-derived lipoproteins [205,238,239]. Lipoprotein uptake by astrocytes has received little attention, probably because the cells are considered to export rather than import lipids. Interestingly, a recent study showed that neurons and astrocytes express distinct splice variants of the VLDL receptor, which differ in their capacity to bind lipoproteins [240]. This observation suggests that the lipoprotein transfer between brain cells is fine-tuned. The relevance of lipoprotein receptors for cholesterol and APOE transport in vivo has been studied in transgenic mice. Complete elimination of LDLR in knockout mice enhances the level of APOE in the CSF by 50% [241], although the cells responsible for this change remained unknown. Neuron-specific ablation of LRP1 using the Cre/loxP system also increased the APOE level and decreased the cholesterol content in the forebrain [242,243]. Conversely, overexpression of a LRP1 fragment with binding activity decreased the APOE level in the brain by 25% [244]. Importantly, neuron-specific ablation of LRP1 causes age-dependent loss of spines and synapses [243] and impairs motor function and memory [232,243]. 4.3. Intracellular redistribution of lipoprotein-derived cholesterol

4.2.1. Expression of lipoprotein receptors in the brain In situ hybridization and immunohistochemical staining revealed that LDLR is expressed by neurons and glial cells [94,214], whereas LRP1 is predominantly expressed by neurons [215–221]. In vitro studies showed that dorsal root ganglion (DRG) cells and hippocampal neurons express more LRP1 than LDLR [222,223], whereas sympathetic neurons expressed LDLR but not LRP1 [224]. At the protein level, cultured astrocytes contained 2-fold more LDLR than LRP1 [223]. Pathologic conditions change the level and cell-specific expression pattern of lipoprotein receptors. In the sciatic nerve, LDLR is expressed at a low level under normal conditions, but strongly upregulated on regenerating axons after injury [225]. Moreover, LRP1 expression has been observed in glial cells including astrocytes in autopsy samples from Alzheimer’s patients [217,220] and following brain injury or neoplastic transformation [226]. Entorhinal cortex lesion in adult rats enhances the density of low density lipoprotein binding sites in granule cells, and in the molecular layer of the dentate gyrus in parallel with astrocyte proliferation [59]. The subcellular distribution of lipoprotein receptors in neurons has been studied in vitro. At a premature stage, LDLR is present in axons and somata of sympathetic [224] and hippocampal neurons with strong expression on growth cones [227]. Studies on compartmented cultures revealed that axons of sympathetic neurons [80] and RGCs [228] take up cholesterol via lipoproteins to support axon regeneration. In mature neurons, LDLR and LRP1 are located in the somatodendritic compartment [229–232]. Notably, LRP1 colocalizes partially with proteins of the postsynaptic density, namely NMDA receptor subunits, which prompted the idea that this receptor is involved in plasticity-related phenomena [232].

Externally acquired cholesterol exits from the endosomal/lysosomal system and reaches subcellular membrane compartments [81–84]. This pathway is relevant in neurons, because dysfunction of two components of late endosomes/lysosomes that mediate the exit of cholesterol, namely Niemann-Pick type C1 (NPC1) and C2 (NPC2), cause neurologic symptoms in patients with Niemann-Pick type C (NPC) disease, an inherited and ultimately fatal human lysosomal storage disorder [14,245–250]. NPC1 is a transmembrane protein with a sterol-sensitive domain [251] and NPC2 is an intralumenal component that binds cholesterol [84,252]. Based on structural data, Kwon and colleagues proposed that NPC2 transfers unesterified cholesterol from endocytosed lipoproteins to NPC1, which then mediates the exit from the endosomal/lysosomal system [253]. The dysfunction of either protein causes accumulation of unesterified cholesterol in the late endosome/lysosome [254– 257]. Mice lacking NPC1 show marked defects in the CNS including formation of meganeurites and ectopic dendrites, axonal degeneration and progressive loss of specific neuronal cell types and glial cells [155,159,258,259], in particular cerebellar Purkinje cells [260,261]. Moreover, the mice show physiopathologic changes in the retina including photoreceptor degeneration, impaired lightevoked responses and changes of the autophagy pathway in RGCs [262]. In situ hybridization and immunohistochemical staining indicated that NPC1 and NPC2 are expressed by both neurons and glial cells [258,263–266]. Accordingly, NPC1 deficiency causes accumulation of unesterified cholesterol and pathologic changes in neurons and glial cells [257,258,267]. Recent studies suggest that the degeneration of neurons in NPC disease is a cell-autonomous

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process: In mice with mosaic expression of NPC1, individual Purkinje cells lacking NPC1 degenerated, whereas those expressing intact NPC1 or those having glial neighbors lacking NPC1 did not [268]. Selective elimination of NPC1 in Purkinje cells in conditional knockout mice was sufficient to induce age-dependent motor deficits and Purkinje cell degeneration [269] and rescue of NPC1-deficiency in neurons prevented neuronal degeneration, whereas rescue in glial cells did not [270]. Notably, NPC1 deficiency did not impair cholesterol secretion by cultured astrocytes [74,173]. Evidence for the opposing view that NPC1 is relevant in glial cells came from the observation that expression of NPC1 under the control of the human GFAP promoter prolongs the life-span of NPC1deficient mice [271]. However, this promoter also drives expression in neuronal precursor cells [141] and thus the rescued lifespan could be due to neuronal expression of NPC1. A possible contribution of glial cells to neurodegeneration and reduced life span in NPC1-deficient mice and humans remains to be defined. Why does NPC1 deficiency cause the cell-autonomous demise of specific neurons? The neurons may depend strongly on the import of external cholesterol via lipoproteins. Support for this idea comes from the appearance of autophagosomes in Purkinje cells [268,272,273] and RGCs of NPC1-deficient mice [262]. These neurons may experience a cholesterol deficit due to the accumulation of cholesterol in the late endosome/lysosome, which they try to compensate by salvaging internal membranes. Alternatively, these neurons may require functions of the endosomal/lysosomal system that are independent from cholesterol import and disturbed by NPC1 deficiency. This includes release and recycling of synaptic vesicles in presynaptic terminals, which is impaired in NPC1-deficient hippocampal neurons in vitro [274–277]. Finally, NPC1 deficiency may cause the demise of neurons by impairing their response to growth factors [278], their mitochondrial function [279] or the structural integrity of axons [280,281]. The cell-spe-

Fig. 4. Neuronal cholesterol homeostasis requires astrocytes. The diagram illustrates the cell-specific distribution of proteins involved in the release, intercellular transport and storage/elimination of cholesterol in neurons (blue) and astrocytes (red). Proteins present in both cell types are indicated in black. The expression patterns suggest that neurons depend on the supply of cholesterol by astrocytes via the LDLR- and LRP1-mediated endocytosis of APOE-containing lipoproteins, and that neurons handle surplus cholesterol by hydroxylation, storage and excretion. Lipoproteins mediating the elimination of cholesterol and 24OH may reach the blood–brain barrier (BBB) via the CSF or astrocytes.

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cific vulnerability of neurons to NPC1 deficiency may depend on high activity of lysosomal enzymes [282]. In summary, there is good evidence that astrocytes secrete cholesterol via APOE-containing lipoproteins, which have distinct properties from those found in blood and which are possibly lipidated by ABC transporters (Fig. 4). The molecular mechanisms of cholesterol remain to be clarified. In vivo and in vitro studies indicate that LDLR and LRP1 mediate uptake of cholesterol- and APOE-containing lipoproteins in CNS neurons and that neuronal cholesterol uptake helps to maintain synaptic integrity (Fig. 4). The age-related degeneration of specific types of neurons lacking NPC1 indicates their strong dependency on an external supply of cholesterol. 5. Handling surplus cholesterol Based on our current knowledge of cholesterol metabolism in neurons and astrocytes, we surmise that only neurons need to deal with cholesterol overload, because they import the component, whereas astrocytes produce cholesterol in excess for its release via APOE-containing lipoproteins. 5.1. Storage after esterification Cells can store surplus cholesterol intracellularly within socalled lipid droplets [283,284]. To this end, cholesterol is esterified by acyl-coenzyme A:cholesterol acyltransferase 1, also called sterol O-acyltransferase 1 (ACAT1/SOAT1), which is located in the ER [285]. Cholesterol esterifying activity was detected in the brains of humans [286], rats [287], chicken [288] and mice [289] at about 1% of the total cholesterol content [289,290]. Transcripts of Acat1/Soat1 are present in the adult mouse brain at region-specific levels, whereas Acat2/Soat2 is expressed in very low amounts except for the thalamus [289,291]. In the rat brain, the concentration of cholesterol esters and the number of Acat1/Soat1 transcripts increase with age [292]. Pharmacologic and genetic reduction of ACAT1/SOAT1 activity in mice decreased the concentration of cholesterol esters in the brain by 86% [293] and ablated cholesterol esterification in homogenates from different brain regions [289], respectively. A genetic association study in humans revealed that a variant of the Acat1/Soat1 gene is associated with lower levels of cholesterol in the CSF [294], which corroborates the idea that ACAT1/SOAT1 activity influences cholesterol metabolism in the CNS. Which cells in the brain show ACAT1/SOAT1 activity? Immunohistochemical staining revealed that the enzyme is present in neurons, but not in glial cells [295]. This is further supported by in situ hybridization data on adult mice, which reveal a complementary distribution of cholesterol esterifying enzymes in the cerebellum, where LCAT is expressed by Bergmann glia, whereas ACAT is expressed by Purkinje cells (Allen Mouse Brain Atlas. Seattle (WA): Allen Institute for Brain Science. Ó2011. Available from: http://mouse.brain-map.org). Experiments on primary cultures showed that the enzyme is active in neurons: Inhibition of ACAT1/SOAT1 in cultured mouse neurons enhanced the ratio of free to esterified cholesterol [296] and incubation of highly purified RGCs with radioactively labeled cholesterol led to intracellular accumulation of cholesterol esters [73]. Notably, cultured astrocytes esterify cholesterol via ACAT1/SOAT1, when they lack APOE and when they are charged with exogenous cholesterol [144]. At present, it remains to be tested, whether neurons or astrocytes store cholesterol in its esterified form. 5.2. Elimination of cholesterol Storage is a possible way to handle surplus cholesterol, but there is good evidence that neurons command specific means for its elimination.

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5.2.1. Conversion to an oxysterol Research within the last decade revealed that neurons employ a unique way to get rid of cholesterol, namely the conversion to 24Shydroxycholesterol (24OHC), which is also called cerebrosterol [7]. The presence of this oxysterol in the human brain has been uncovered more than 50 years ago [297], but its function has remained obscure until its implication in cholesterol elimination from the brain was discovered. Pioneering work by the Bjorkhem group revealed synthesis of 24OHC in the brain in vivo, a continuous flux from the brain to blood and its elimination as bile [50,298–301]. Moreover, the group showed that the concentration of 24OHC in the CSF and blood declines with age [298]. Hydroxylation of cholesterol at C24 strongly accelerates its translocation between lipophilic compartments and thereby facilitates the passage across the blood–brain barrier [302]. A recent study showed that genetic elimination of ACAT1/SOAT1 in a triple transgenic mouse model of Alzheimer’s disease decreases cholesterol synthesis and enhances the 24OHC level in the brain [289]. These findings suggest that if neurons loose the ability to store cholesterol as esters, they eliminate excess amounts by conversion to 24OHC. There is evidence that 24OHC plays a dual role. Besides being metabolite for elimination of cholesterol, it may act as a signaling molecule that regulates cholesterol homeostasis [303] and developmental processes like neurogenesis [304] by activating LXRs. Neuron-derived 24OHC may signal to astrocytes the neuronal cholesterol levels. Such a dual role raises the question, whether the intra- and intercellular transport of 24OHC is purely diffusion-driven, or whether there are mechanisms for a targeted transport. Kim and colleagues showed that 24OHC release from cultured neurons is enhanced by the presence of APOE [305] suggesting that the oxysterol hitchhikes on lipoproteins. A pharmacologic study in adult rats in vivo indicated that the transport of 24OHC across the blood–brain barrier is mediated by an organic anion transporter [306]. The intracellular transport of 24OHC may involve oxysterol-binding proteins [307]. These components are expressed in the brain [308], but their functions in neurons or astrocytes remain to be unexplored. The formation of 24OHC is catalyzed by a member of the cytochrome P450 family, cholesterol 24-hydroxylase (CYP46A1) [309]. This enzyme is mainly found in the gray matter of the CNS and its level increases steadily during postnatal development to reach a constant plateau in the adult [309,310]. Immunohistochemical staining showed its expression in neurons of different regions of the embryonic and adult rodent brain including cerebellar cortex, thalamus, hippocampus and retina [309,311,312], but not by astrocytes [311]. Interestingly, the staining intensity differs among neuronal cell types with large neurons like pyramidal cells, Purkinje cells and RGCs showing high levels of expression. This supports the idea that the rate of cholesterol turnover differs among neurons [313]. Immunocytochemical staining of cultured neurons revealed that CYP46A1 is located in the ER and present in somata and dendrites, but not in axons or presynaptic terminals [311]. Several studies suggest that brain injury or disease shift CYP46A1 expression from neurons to microglia [314] or astrocytes [315–318], but the functional relevance remains unclear. The promoter region of Cyp46a1 contains binding sites for transcription factors that regulate housekeeping enzyme and shows relative insensitivity to gene regulatory pathways [310,319]. These observations indicate that neurons express Cyp46a1 at a fairly constant level. Neuronal synthesis of 24OHC from radioactively labeled cholesterol and its secretion were confirmed in primary cultures of fetal human neurons [305] and of highly purified RGCs from postnatal rats [73]. The latter study also confirmed exclusive expression of CYP46A1 in neurons, but not astrocytes. Further hydroxylation of 24OHC at C25 has been observed in cultured neurons and astrocytes [320,321], but the relevance of this compound remains unknown.

The physiologic importance of 24OHC was revealed by CYP46A1 knockout mice. These mice develop normally, but show strongly reduced levels of 24OHC in the brain, which confirmed ablation of enzymatic activity [322]. The rate of cholesterol synthesis in the brain of CYP46A1 knockout mice is reduced by 40% probably to compensate for the reduced elimination. This relatively strong reduction indicates that CYP46A1-expressing neurons command a considerable fraction of cholesterol turnover in the adult brain [322,323]. It was also shown that CYP46A1 deficiency in mice reduces the efflux of cholesterol from the brain by two thirds [323]. This important finding points to CYP46A1-dependent and independent modes of cholesterol excretion from the brain. Further evidence for a CYP46A1-independent pathway comes from a thorough quantitative analysis of cholesterol accretion and synthesis rates in mice. This study showed that pharmacological activation of LXRs in adult mice enhanced cholesterol excretion from the brain independently from an increase in transcript levels of Cyp46a1 [324]. 5.2.2. Secretion via ABC transporters A CYP46A1-independent pathway for cholesterol elimination from neurons may be the direct release onto APOA1-containing lipoproteins that are present in the CSF [93,94,140]. If neurons employ such a mode of release, they must express ABC transporters that mediate cellular sterol efflux, namely ABCA1, ABCG1 and ABCG4 [188]. In situ hybridization and immunohistochemical staining revealed that ABCA1 is expressed by neurons in embryonic and adult rodents [189–191,262,325], with high expression levels in large cells like hippocampal pyramidal cells and Purkinje cells [190,191]. The regional and cell-specific distribution of ABCG1 and ABCG4 has been determined using knock-in mice, which express beta-galactosidase under the control of the endogenous Abcg1 and Abcg4 promoters [326]. The studies showed strong neuronal expression of both transporters in the embryonic and adult brain and their colocalization in specific neuronal cell types like hippocampal pyramidal cells, cerebellar Purkinje cells and RGCs [75,192,193]. Studies on primary cultures revealed that basal levels of ABCA1, ABCG1 and ABCG4 are five times higher in neurons compared to astrocytes [192] and that pharmacologic activation of the LXR pathway induces an increase in mRNA levels of ABCG1, but not of ABCG4 in neurons and astrocytes [192,204]. The subcellular distribution of the transporters has been studied in primary cultures and cell lines by immunocytochemical staining and by transfection with tagged proteins. ABCA1 was present in perinuclear compartments of neurons and on the surface of axons, somata and dendrites [189,191], whereas ABCG1 and ABCG4 colocalized in endosomal vesicles, which were predominantly present in axons [192]. Do ABC transporters mediate cholesterol efflux from neurons? So far, answers come solely from in vitro studies. Treatment of primary neuronal cultures from embryonic rats with an LXR agonist, enhanced ABCA1 expression and, with the same time course, cholesterol efflux onto externally applied apolipoproteins [191]. In an earlier paper, Whitney and colleagues did not observe a LXR-induced increase in cholesterol efflux from cultured neurons even in the presence of external apolipoproteins, although the treatment increased Abca1 and Abcg1 transcripts [194]. The divergent results are probably due to different experimental protocols. A more direct proof that ABCA1 mediates neuronal cholesterol efflux was provided recently [327]. Downregulation of ABCA1 levels by RNA interference in cultured neurons reduced the efflux of cholesterol and phospholipids onto externally supplied APOE, whereas pharmacologic enhancement of ABCA1 levels enhanced the lipid release [327]. So far, there is no direct evidence that neurons excrete cholesterol in vivo. Pharmacologic enhancement of ABCA1 and ABCG1 levels in mice increases cholesterol excretion from the brain by

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1.6-fold [324], but the cellular source remains to be defined. Cellspecific elimination or overexpression of ABC transporters remains to be accomplished. The idea that neurons dispose of cholesterol directly via preformed APOA1-containing lipoproteins raises the question, how these lipoproteins are then eliminated from the brain. This could involve LRP1 or scavenger receptor class B1, which are both expressed by brain capillary endothelial cells [328,329]. LRP1 binds APOE, which may also be present on these lipoproteins, and mediates the clearance of beta-amyloid from the brain [330]. SCARB1 has been shown to promote efflux of cholesterol via brain capillary endothelial cells in vitro [328]. The APOA1-containing lipoproteins carrying surplus cholesterol from neurons may pass through astrocytes, which express SCARB1 [331–333], and whose endfeet are in touch with the capillary endothelial cells (Fig. 4). We note that absence of SCARB1 in mice does not affect sterol turnover or APOE levels [57,334], but may induce age-related synaptic defects and memory deficits [335]. Taken together, neurons may handle excess cholesterol by esterification and subsequent intracellular storage, by direct excretion via ABC transporters and by conversion to 24OHC (Fig. 4). 6. Conclusion and outlook The investigation of cholesterol metabolism in the brain has a long history, but it is only within the last decade that this field has gained momentum (Fig. 5), probably because cholesterol is implied in neurodegenerative disease. It is evident that cells in the CNS employ their proper system to maintain their cholesterol content, which is very different from the way cholesterol metabolism is organized in the rest of the body. Our focus on neurons and astrocytes, whose cooperation is necessary for brain development and function, exposes several peculiarities. First, there is a remarkable cell-specific distribution of proteins that are involved in cholesterol metabolism (Figs. 3 and 4). For example, astrocytes, but not neurons, express APOE and secrete lipoproteins. On the other hand, neurons, but not astrocytes, express CYP46, which enables removal of surplus cholesterol from the brain. Second, cell-specific gene ablation in transgenic mice revealed that neurons must synthesize cholesterol to survive during early development, but not in the adult, and that neuronal lipoprotein uptake affects APOE and cholesterol levels and is necessary for spine integrity and brain function. Notably, there is evidence that several pathways mediate cholesterol excretion from the brain, one of which is cholesterol hydroxylation by neurons. Third, in vitro studies revealed that

Fig. 5. Publications on cholesterol in selected organs during postwar decades. Number of publications during indicated periods normalized to paper counts during the last interval. The numbers of publications were extracted from boolean PubMed searches on cholesterol and the indicated organ. Numbers in legend indicate paper counts during last decade and the total number of publications. Note the stagnation of research on cholesterol in the brain during the 70s and 80s, and the 2-fold increase within the last decade. Based on total paper counts, research on brain represents a smaller field compared to studies on blood or the liver.

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astrocytes and neurons synthesize cholesterol by different pathways and at different rates. Together, these observations insinuate that cholesterol turnover in neurons depends on close metabolic cooperation with astrocytes. New questions arise. For example, how high is the cholesterol turnover in neurons during development and in the adult? Do all neurons rely on cholesterol from astrocytes? How do axons, dendrites and synapses build and maintain their cholesterol content? Where and how is the transport of cholesterol from astrocytes to neurons accomplished? Does the intra- and intercellular transport of oxysterols involve dedicated carriers? Why are components of cholesterol metabolism upregulated after injury and disease? Answering these questions requires new experimental approaches to map the cellular and subcellular distribution of cholesterol and of proteins involved in its homeostasis and new mouse models to monitor and interfere with cholesterol metabolism in a cell-specific manner. Whatever lies ahead in this exciting field, we predict that an understanding of cholesterol metabolism in the brain and its role in disease requires meticulous study of individual cell types in the CNS. Acknowledgements The authors gratefully acknowledge previous and ongoing support by Abbott/Laboratoires Fournier, Agence Francaise Contres les Myopathies, Agence National de la Recherche, Ara Parseghian Medical Research Foundation, Centre National de la Recherche Scientifique, Deutsche Forschungsgemeinschaft, ELTEM, European Commission Coordination Action ENINET (contract number LSHM-CT-2005-19063), Fondation NRJ-Institut de France, Fondation pour la Recherche Medicale, Max-Planck Society, Merck-Serono, Miltenyi Biotec, Ministère de l’Enseignement Supérieur et de la Recherche, Neurex, Région Alsace, Retina France, Sanofi-Aventis and Université de Strasbourg. References [1] Linton MF, Gish R, Hubl ST, Butler E, Esquivel C, Bry WI, et al. Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation. J Clin Invest 1991;88:270–81. [2] Dietschy JM, Turley SD. Thematic review series: brain lipids. Cholesterol metabolism in the central nervous system during early development and in the mature animal. J Lipid Res 2004;45:1375–97. [3] Camargo N, Smit AB, Verheijen MH. SREBPs: SREBP function in glia–neuron interactions. FEBS J 2009;276:628–36. [4] De Chaves EP, Narayanaswami V. Apolipoprotein E and cholesterol in aging and disease in the brain. Future Lipidol 2008;3:505–30. [5] Dietschy JM. Central nervous system: cholesterol turnover, brain development and neurodegeneration. Biol Chem 2009;390:287–93. [6] Mulder M. Sterols in the central nervous system. Curr Opin Clin Nutr Metab Care 2009;12:152–8. [7] Russell DW, Halford RW, Ramirez DM, Shah R, Kotti T. Cholesterol 24hydroxylase: an enzyme of cholesterol turnover in the brain. Annu Rev Biochem 2009;78:1017–40. [8] Vance JE, Hayashi H. Formation and function of apolipoprotein E-containing lipoproteins in the nervous system. Biochim Biophys Acta 2010;1801:806–18 [9] Ikonen E. Mechanisms for cellular cholesterol transport: defects and human disease. Physiol Rev 2006;86:1237–61. [10] Valenza M, Cattaneo E. Cholesterol dysfunction in neurodegenerative diseases: is Huntington’s disease in the list? Prog Neurobiol 2006;80:165–76. [11] Carter CJ. Convergence of genes implicated in Alzheimer’s disease on the cerebral cholesterol shuttle: APP, cholesterol, lipoproteins, and atherosclerosis. Neurochem Int 2007;50:12–38. [12] Hirsch-Reinshagen V, Wellington CL. Cholesterol metabolism, apolipoprotein E, adenosine triphosphate-binding cassette transporters, and Alzheimer’s disease. Curr Opin Lipidol 2007;18:325–32. [13] Bu G. Apolipoprotein E and its receptors in Alzheimer’s disease: pathways, pathogenesis and therapy. Nat Rev Neurosci 2009;10:333–44. [14] Karten B, Peake KB, Vance JE. Mechanisms and consequences of impaired lipid trafficking in Niemann-Pick type C1-deficient mammalian cells. Biochim Biophys Acta 2009;1791:659–70. [15] Martins IJ, Berger T, Sharman MJ, Verdile G, Fuller SJ, Martins RN. Cholesterol metabolism and transport in the pathogenesis of Alzheimer’s disease. J Neurochem 2009;111:1275–308. [16] Fliesler SJ, Bretillon L. The ins and outs of cholesterol in the vertebrate retina. J Lipid Res 2010;51:3399–413.

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