Hong Wang and Robert H. Eckel
Lipoproteins in the brain are mostly made in the brain.
Lipoprotein composition in the brain is different from that in the circulation.
Neurons and astrocytes coordinate lipoprotein metabolism in the brain.
Lipoproteins help to regulate neurobehavioral function and energy balance.
Lipoproteins in plasma transport lipids between tissues, however, only high-density lipoproteins (HDL) appear to traverse the blood–brain barrier (BBB); thus, lipoproteins found in the brain must be produced within the central nervous system. Apolipoproteins E (ApoE) and ApoJ are the most abundant apolipoproteins in the brain, are mostly synthesized by astrocytes, and are found on HDL. In the hippocampus and other brain regions, lipoproteins help to regulate neurobehavioral functions by processes that are lipoprotein receptor-mediated. Moreover, lipoproteins and their receptors also have roles in the regulation of body weight and energy balance, acting through lipoprotein lipase (LPL) and the low-density lipoprotein (LDL) receptor-related protein (LRP). Thus, understanding lipoproteins and their metabolism in the brain provides a new opportunity with potential therapeutic relevance.
Lipoprotein metabolism in the systemic circulation
Plasma lipoproteins contain lipids and proteins, and transport lipids in a polar environment. Four major classes of lipids are found in lipoproteins: triacylglycerols (TGs), unesterified cholesterol, cholesteryl esters (CE), and phospholipids. Being the most polar, phospholipids are associated with apolipoproteins and surround the less polar lipids. Apolipoproteins serve as enzyme cofactors and receptor ligands.
Lipid delivery by lipoproteins and their processing are regulated by lipoprotein receptors located on cell surfaces such as the LDL receptor (LDLR) and LRP, as well as other proteins associating with lipoproteins, for example lipoprotein-associated phospholipase A2 (LpPLA2) and serum amyloid A (SAA). Recent evidence also suggests that circulating extracellular microRNAs (miRNAs) are associated with lipoproteins for their transfer between cells .
Lipoproteins are traditionally classified by density. The larger lipoprotein particles – chylomicrons and very low density lipoproteins (VLDL) are the TG-rich lipoproteins produced by the intestine and liver. LDLs are predominantly formed by the hydrolysis of TG-rich lipoproteins by LPL. HDLs are also secreted by the intestine and liver as nascent ApoA-I/phospholipid discs which incorporate cholesterol through the ATP-binding cassette transporter proteins. HDL increases in lipid content and size when TG-rich lipoproteins are processed by LPL, and facilitates reverse cholesterol transport for biliary excretion. Lipoprotein metabolism has been well characterized in plasma, but much less is known about lipoproteins in the brain.
Major differences between lipoproteins in plasma versus in the brain
Early studies of lipoproteins in brain focused on particles in the cerebrospinal fluid (CSF) [2, 3, 4]. Using gel electrophoresis and electron microscopy, CSF lipoproteins were shown to be mostly spherical, resembling the size and density of plasma HDL . Unlike plasma, the most abundant apolipoprotein in CSF lipoproteins is ApoE, which is usually localized to the largest particles . ApoA-I and ApoA-II are present on smaller particles, and ApoJ is distributed across the particle size-range. Other apolipoproteins such as ApoA-IV, ApoD, and ApoH are also found in the CSF. In addition, some CSF lipoproteins are found to be associated with amyloid beta (Aβ, suggesting a role for lipoproteins in Aβ polymerization, transport, and clearance . Another source of lipoproteins in the CSF is the choroid plexus. For example, ApoB-containing lipoproteins, which can be identified in CSF, are found at a concentration consistent with porous diffusion enhanced by CSF secretion [5, 6].
Despite the existence of the BBB, some of the smaller circulating HDL lipoproteins can enter the brain [2, 4]. However, when mice are injected with adenovirus expressing human ApoE isoform 3 (ApoE3), ApoE3 protein is found in plasma lipoproteins at high levels but remains undetectable in CSF . In brain, ApoE is expressed predominantly by astrocytes and microglia, and in reduced quantity in neurons, whereas ApoJ is expressed in astrocytes, neurons, and the ependymal cells lining the ventricle . The majority of the ApoE- and ApoJ-containing lipoproteins found in CSF are thought to originate from surrounding astrocytes.
Cholesterol is the most-studied lipid in the brain. In the brain, the BBB necessitates that cholesterol homeostasis be maintained by local synthesis, and this process has been studied in detail in mice . Cholesterol transporter Niemann–Pick type C protein 1 and cholesterol 24(S)-hydroxylase are essential for cholesterol metabolism in the brain, although changes in the plasma cholesterol concentration or loss of function of ATP-binding cassette AI transporter (ABCA1), scavenger receptor class B, type I (SR-B1, also known as Scarb1), LDLR or ApoE, or ApoA-I have no effect on sterol turnover in the brain . Overall, data strongly suggest that during early development, cholesterol originates entirely from local synthesis, but in the adult there is a constant excretion of sterol from the brain into the plasma .
Lipoprotein synthesis, assembly, and metabolism in astrocytes and other cell types in the brain
Astrocytes are specialized star-shaped glial cells that surround and support neurons to provide essential nutrients for neuronal growth and function. Astrocytes are also considered to be the major sites for synthesis of lipoprotein constituents and lipoprotein assembly in the brain. However, recent evidence suggests that, at least for cholesterol, astrocytes and neurons cooperate in the regulation of its synthesis and redistribution in the brain . Specifically, selected enzymatic steps and precursors in the biosynthesis of cholesterol differ in cultured astrocytes versus neurons. In addition, different mechanisms appear to regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles these cell types play in brain cholesterol homeostasis. For example, astrocytes produce and release ApoE, whereas neurons metabolize cholesterol to 24(S)-hydroxycholesterol.
Cholesterol efflux from astrocytes is facilitated by apolipoproteins alone or lipoprotein particles, whereas cholesterol removal from neurons is triggered only by lipoprotein particles. ABCA1- and ABCG1-regulated cholesterol efflux occurs only in astrocytes whereas ABCG4-mediated cholesterol efflux takes place only in neurons . Furthermore, the newly synthesized cholesterol is rarely converted to CE, and is quickly redistributed among various cell types within the brain (reviewed in ). Moreover, the half-life of brain-derived cholesterol is much longer (up to 5 years) compared to that of days in the periphery, with extensive redistribution and transportation via ATP-binding cassette transporters and HDL-like lipoproteins respectively, as the main mechanism to maintain homeostasis.
Apolipoproteins are expressed at higher levels in astrocytes than the rest of the brain. The mRNA and protein levels of ApoE and ApoJ are age-dependent – with ApoJ increasing ∼5–10-fold and ApoE levels dropping with aging . ApoJ is a ubiquitous multifunctional glycoprotein and its expression in the brain is upregulated in response to neuronal damage, brain injury and other stress; and ApoJ has been proposed to play a role in Aβ clearing . ApoJ-containing lipoprotein particles usually contain the least amount of lipid, whereas ApoE-containing lipoproteins carry the most lipid and are the largest in size .
The role of ApoE-containing lipoproteins in the brain has been extensively studied and reviewed. In brief, three major functions have been suggested for astrocyte-derived ApoE-containing lipoproteins: (i) the transfer of phospholipids and cholesterol via ATP-binding cassette (ABC) transporters such as ABCA1 and ABCG1 ; (ii) interaction with the LDLR superfamily of proteins located on the surface of neurons to facilitate axonal growth and neuronal survival ; and (iii) interaction with the LRP1-dependent cellular uptake pathway in the deposition of amyloid plaques [16, 17]. Although there is minimal direct interaction between ApoE and soluble Aβ in CSF , ApoE isoforms in ApoE-containing lipoprotein complexes can regulate the metabolism of soluble Aβ by competing for the binding of LRP1 with Aβ in astrocytes [18, 19].
ApoE knockout mice placed on a diet enriched in homocysteine to induce oxidative stress, show impaired learning and memory . Of the three major isoforms of ApoE – ApoE2, ApoE3, and ApoE4 – ApoE4 confers the major risk for Alzheimer’s disease (AD). The expression of the ApoE4 allele usually results in increased expression of ApoC1 . Mice overexpressing human ApoC1 also display impaired learning and memory . Interestingly Apoc1−/− mice also show impaired hippocampal-dependent memory with no gross changes in brain morphology or brain cholesterol levels, but increased expression of the proinflammatory marker tumor necrosis factor-α .
With all the evidence discussed above several major questions remain: (i) what is the role of neurons in the synthesis and regulation of lipoprotein metabolism? (ii) Can different lipoprotein particles enter neurons or be recognized by surface markers on neurons? (iii) What are the major functions of lipoproteins in the brain – is it simply lipid delivery and/or are they carriers for various biological molecules? Some of the answers to these questions may relate to the existence and properties of specific lipid structures in the brain, and the cell type- and region-specific expression of lipoprotein receptors in the brain.
Lipid rafts and neuronal porosomes
Lipid rafts are cholesterol-enriched domains in biomembranes that serve as the preferential clustering site of membrane signaling proteins. Lipid rafts exist in both neurons and astrocytes . In addition to cholesterol and sphingolipids, saturated fatty acids are enriched in lipid rafts. In systemic circulation, lipid rafts serve as a signaling platform linking lipoprotein metabolism to atherosclerosis . In neurodegenerative diseases, lipid raft disarrangement may be an early marker for diagnosis . Size-exclusion chromatography and electron microscopy have been used to study the lipid composition of nascent HDL formed by ABCA1. The proportions of free cholesterol, glycerophosphocholine, and sphingomyelin are similar between nascent HDL and lipid rafts , suggesting a possible role of lipid rafts in lipoprotein assembly in addition to their role in facilitating lipoprotein-mediated lipid exchange.
Porosomes are universal secretory portals at the plasma membrane that facilitate the transient docking and fusion of membrane-bound secretory vesicles to exchange intravesicular contents to the outside . In neurons, 12–17 nm cup-shaped porosome structures are present at the presynaptic membrane where 40–50 nm synaptic vesicles transiently dock and fuse to release neurotransmitters . The neuronal porosome complex has been isolated, its composition determined, and it has been structurally and functionally reconstituted in artificial lipid membranes [30, 31]. Cholesterol was found to be an integral component of the neuronal porosome complex, and crucial to the stability of the porosome/fusion pore [32, 33]. Porosomes are also found in astrocytes and have a similar structure but are smaller in size (10–15 nm) [34, 35]. Proteomic analysis of neuronal porosomes revealed the absence of any typical lipoprotein-associated proteins, but this does not exclude the possibility that porosomes can serve as a platform for lipoprotein-mediated lipid exchange in the brain.
Lipoprotein receptors in the central nervous system (CNS)
The LDLR superfamily of proteins are a class of single membrane-spanning domain receptors that bind ApoB100 or ApoE and also endocytose a variety of distinct extracellular proteins. In peripheral tissues, the tissue- or cell type-specific presence of different lipoprotein receptors dictates where lipoprotein particles will be docked and lipoprotein-mediated lipid exchange will occur. For example, HDL binds to a different set of cell surface receptors, some for cholesterol efflux (i.e., ABCA1 and ABCG1) and others for uptake and degradation (i.e., SR-B1). Some of these receptors are also expressed in the brain.
In the periphery, SR-B1 mediates the selective uptake of HDL-associated CE independently from HDL internalization. In the brain, SR-B1 is expressed in endothelial cells and contributes to the selective uptake of HDL-associated CE and vitamin E (α-tocopherol), once HDL traffics through the BBB [36, 37, 38]. The importance of SR-B1 in regulating systemic and brain vitamin E metabolism is supported by the observation that SR-B1-deficient mice display higher plasma levels of α-tocopherol accompanied by decreases in α-tocopherol in several tissues and the brain . Together with ABCA1 and ApoA-1, SR-B1 can modulate the polarized sterol mobilization at the BBB . SR-B1 in the brain also appears to mediate phosphatidylcholine uptake from LDL or HDL, resulting in increases in fatty acid content . This process provides a route for generating a pool of polyunsaturated fatty acid (PUFA)-containing lipids for transport into deeper brain regions.
LDLR knockout (Ldlr−/−) mice show no major deficits in sensory or motor function, but exhibit increased locomotor activity . Ldlr−/− mice also show a decrease in learning and memory regardless of diet . Cholesterol-enriched diets increase plasma cholesterol levels, which relate to an increase in oxidative stress, decrease in the mitochondrial complex I and II activities in the cerebral cortex, and cognitive impairment . In aged Ldlr−/− mice that are exposed to cholesterol-enriched diets earlier in life, antioxidant imbalance and oxidative damage are evident by a marked increase in lipid peroxidation and an increase in acetylcholinesterase activity in the prefrontal cortex. Both working and spatial preference as well as procedural memory are impaired, without alterations in motor function .
Very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2)
VLDLR binds TG-rich lipoproteins but not LDL, and, in the periphery, also serves as a remnant lipoprotein receptor. The VLDLR in various tissues usually functions in concert with LPL. In the brain, various mutations in VLDLR have been associated with disequilibrium syndrome (DES) [44, 45], autosomal recessive cerebellar hypoplasia leading to mental retardation , and quadrupedal locomotion in humans . In developing brain, VLDLR plays a role in neuronal migration together with ApoER2 (also known as LDLR-related protein 8, LRP8), a cell surface receptor and a member of the LDLR family, with 50% sequence homology to VLDLR. VLDLR/ApoER2 double knockout mice have a disruption of cortical layering and cerebellar dysmorphology . VLDLR knockout mice alone display contextual fear conditioning deficits and a moderate defect in long-term potentiation (LTP) .
ApoER2 knockout mice display similar deficits in contextual fear conditioning and a more pronounced defect in LTP , and decreased dendritic spine density in cortical layers II/III , but cholesterol and PUFA concentrations throughout the brain are not different . An earlier study using radiolabeled fatty acids incorporated into lipoproteins suggests indirectly that circulating fatty acids within VLDLs do not enter the brain in the awake rodent . These data suggest that deficits in Vldlr−/− and ApoER2 (Lrp8−/−) knockout mice are more related to the reelin-dependent pathway that regulates processes of neuronal migration and positioning, and synaptic plasticity through modulation of N-methyl-D-aspartic acid (NMDA) receptor activity [49, 53] in complex with the post-synaptic density protein PSD-95  in the brain.
LRP1 is an endocytic receptor that mediates the cellular uptake of various ligands including chylomicron remnants. In brain, LRP1 is expressed at high levels in both glial and neuronal cells to mediate endocytosis of ligands, regulate calcium influx into neurons after stimulation with NMDA, interact with amyloid precursor protein (APP) to regulate Aβ clearing, and interact with PSD-95 to regulate synaptic transmission [54, 55, 56, 57, 15]. LRP1 forebrain knockout mice (using CaMKII-driven Cre) display impaired brain lipid metabolism with decreased cholesterol and TG in cortex, an age-dependent decrease in spine density in pyramidal neurons in the cortex and CA1 region in the hippocampus, and progressive neurobehavioral abnormalities characterized by impaired memory, motor function, and reduced LTP .
Novel roles of lipoproteins in the CNS: lipoprotein metabolism in the neurons
Many of the lipoprotein receptors discussed above are expressed in both astrocytes and neurons. Even though astrocytes are considered to be the major cells mediating larger lipoprotein assembly, increasing evidence suggests that lipoprotein components synthesized by neurons might be important in regulating lipoprotein metabolism in the brain. Data from neuron-specific modification of LRP1 discussed above are in line with such hypothesis.
With the recent development of the ApoE knockout and knock-in mouse models, it is feasible to relate the ApoE protein made in different cell types in the brain to its potential unique biological activities . ApoE knock-in mice have been generated on a background of Apoe−/− mice to express human ApoE isoforms either in astrocytes or neurons. Expression of the human ApoE3 isoform protects the neuronal synapses and dendrites from the excitotoxicity seen in ApoE-deficient mice. Astrocyte-derived ApoE4 displays similar protective effects to ApoE3, whereas neuronal expression of ApoE4 results in loss of cortical neurons after an excitotoxic challenge . Neuronal ApoE4 knock-in mice also display more severe and widespread deficits in dendritic arborization, as well as in spine density and morphology, than do astrocyte ApoE4 knock-in mice . The cellular source of ApoE also impacts upon its role in the clearance of Aβ. During the Aβ clearing process, astrocyte-derived ApoE becomes drained into the perivascular space (PVS) whereas neuronal ApoE4 does not, and, when these knock-in mice are crossed with APP transgenic mice, both neuronal and astrocyte-derived ApoE4 are found to be colocalized in the PVS, and astrocytes take up the neuronal ApoE4 bound to Aβ, but not neuronal ApoE4 alone .
Recent studies in neuron-specific Lrp1−/− mice suggest another role for lipoprotein metabolism in the CNS. LRP1 forebrain knockout mice display increased food intake, decreased energy consumption, and decreased leptin signaling, resulting in obesity . Direct injection of Cre-expressing lentivirus into the arcuate nucleus of the hypothalamus of Lrp1 Flox-P mice that leads to region-specific neuronal Lrp1 deletion resulted in increased food intake, markedly increased NPY and AgRP gene expression, and greater body weight gain. Neuronal inactivation of LRP1 via the synapsin-driven Cre also leads to increases in food intake, but these mice die prematurely at between 6–9 months of age as a result of hypercatabolism .
The potential role of lipoprotein-derived lipid in the CNS regulation of energy balance is most strongly supported by data from a neuron-specific LPL-deficient mouse model . Deficiency of LPL, the rate-limiting enzyme in the hydrolysis of TG-rich lipoproteins in the periphery  in neurons increases food intake, reduces energy expenditure, upregulates neuropeptide AgRP and NPY gene expression, reduces PUFA concentrations in TG and FFA pools in the hypothalamus, reduces TG-rich lipoprotein-derived lipid uptake in the hypothalamus, and results in obesity even on a chow diet . LPL mRNA expression is detected in many regions of the brain, and in both neurons and astrocytes, with the hippocampal area having the highest levels of expression . Very interestingly, general LPL knockout mice rescued from neonatal lethality by somatic gene transfer (no exogenous or endogenous LPL expression in the brain) display impaired learning and memory function compared to other neuron-specific or general knockout mouse models of many lipoprotein receptors described earlier . These neuron-specific LPL-deficient mice also show reductions of the presynaptic marker synaptophysin (instead of the postsynaptic marker PSD95) in the hippocampus, a decreased number of presynaptic vesicles, and reductions of vitamin E content in the brain .
Phenotypes related to the specific functions of lipid-related genes in the brain are summarized in Table 1. Based on the data accrued from these genetically-modified mice and the data from ex vivo experimentation discussed above, a number of common themes emerge: (i) Most of the lipoprotein receptors that recognize larger particles (LDLR, VLDLR, ApoER2, and LRP1) relate to particular types of neurobehavioral functions. (ii) Only the traditional HDL receptor SR-B1 and the LRP1 seem to modify brain cholesterol, TG, and vitamin E content. (iii) Although the lipoprotein receptors for larger lipoproteins are not essential for maintaining brain lipid content, various knockout mice do display phenotypes in response to dietary perturbations, implying a potential link between lipoprotein metabolism in the CNS and dietary lipids.
|Gene modified||Loss or gain of function||Cell- or tissue-specificity||Brain phenotype||Behavioral phenotype||Notes||Refs|
|Apoe||Loss||Whole body||↓ Synapses and dendrites ↑ excitotoxicity||↓ Learning and memory||On a homocysteine-enriched diet|||
|Apoe3 in Apoe−/−||Replacement||Whole body||Protects against ↓ neuronal synapses and dendrites from excitotoxicity|||
|Apoe4 in Apoe−/−||Replacement||Astrocyte||Protects against ↓ neuronal synapses and dendrites from excitotoxicity|||
|Apoe4 in Apoe−/−||Replacement||Neuron||↓ Cortical neurons after excitotoxic challenge; more severe ↓ in dendritic arborization, spine density, and morphology|||
|Apoc1||Gain||Whole body||↓ Learning and memory|||
|Apoc1||Loss||Whole body||↓ Learning and memory|||
|Loss||Whole body||↑ Brain
|Ldlr||Loss||Whole body||↑ Lipid peroxidation
↑ Oxidative stress
|↓ Learning and memory
↑ Locomotor activity
|Cholesterol-enriched diet for metabolic phenotypes||[41, 42, 43]|
|Vldlr||Loss||Whole body||↓ Contextual fear conditioning
Moderate ↓ in long-term potentiation (LTP)
|Loss||Whole body||↓ Dendritic spine density in cortical layers II/III||Contextual fear
Moderate ↓ in LTP
|[49, 50, 51]|
|Vldlr/ApoER2||Loss||Whole body||Disruption of cortical layering and cerebellar dysmorphology|||
|Lrp1||Loss||CaMKII-driven Cre, forebrain||Obesity, ↑ food intake||↓ Memory, motor function, and
|↓ Cholesterol and TG in cortex||[58, 63]|
|Lrp1||Loss||Synapsin-driven Cre||↑ Food intake||Died earlier due to hypercatabolism|||
|Lrp1||Loss||Hypothalamus||↑ Food intake, obesity||↑ AgRP and NPY levels|||
|Lpl||Loss||Whole body rescued||↓ Learning and memory function|||
|Lpl||Loss||Neuron||↑ Food intake, obesity||↓ PUFA
↑ AgRP, NPY
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