Neuronal Lipid Metabolism: Multiple Pathways PDF

REVIEW published: 23 January 2018...

REVIEW published: 23 January 2018 doi: 10.3389/fnmol.2018.00010 Neuronal Lipid Metabolism: Multiple Pathways Driving Functional Outcomes in Health and Disease Timothy J. Tracey 1 *, Frederik J. Steyn 2 , Ernst J. Wolvetang 1 and Shyuan T. Ngo 1,2,3 * 1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia, 2 Centre for Clinical Research, The University of Queensland, Brisbane, QLD, Australia, 3 Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia Lipids are a fundamental class of organic molecules implicated in a wide range of biological processes related to their structural diversity, and based on this can be broadly classified into five categories; fatty acids, triacylglycerols (TAGs), phospholipids, sterol lipids and sphingolipids. Different lipid classes play major roles in neuronal cell populations; they can be used as energy substrates, act as building blocks for cellular structural machinery, serve as bioactive molecules, or a combination of each. In amyotrophic lateral sclerosis (ALS), dysfunctions in lipid metabolism and function have been identified as potential drivers of pathogenesis. In particular, aberrant lipid metabolism is proposed to underlie denervation of neuromuscular junctions, mitochondrial dysfunction, excitotoxicity, impaired neuronal transport, cytoskeletal defects, inflammation and reduced neurotransmitter release. Here we review current knowledge of the roles of lipid metabolism and function in the CNS and discuss how Edited by: Jean-Philippe Loeffler, modulating these pathways may offer novel therapeutic options for treating ALS. Institut National de la Santé et de la Recherche Médicale, France Keywords: lipid metabolism, neuronal metabolism, amyotrophic lateral sclerosis, mitochondria, glycosphingolipid Reviewed by: Mauro Cozzolino, Istituto di Farmacologia Traslazionale INTRODUCTION (CNR), Italy Alberto Ferri, Lipids are fundamental organic molecules that are utilized by the human body for a number of Istituto di Biologia Cellulare e Neurobiologia (CNR), Italy essential cellular processes. Broadly speaking, lipids can be classified into five major subcategories. These include fatty acids, triglycerides, phospholipids, sterol lipids and sphingolipids. Significant *Correspondence: Timothy J. Tracey diversity in lipid structure exists, with between 1000 and 2000 different lipid species thought to [email protected] exist in mammalian biological systems. The importance of lipids to biological systems is highlighted Shyuan T. Ngo by the fact that 5% of all human genes are devoted to lipid synthesis (van Meer et al., 2008). The [email protected] brain makes great use of all five classes of lipids, and contains the second highest concentration of lipids in the human body (Hamilton et al., 2007). In this review, we discuss their individual Received: 26 November 2017 structure, synthesis, and transport in the context of brain function. We then discuss their roles in Accepted: 08 January 2018 Published: 23 January 2018 amyotrophic lateral sclerosis (ALS) pathogenesis and discuss how modulating lipid function may offer novel therapeutic options. Citation: Tracey TJ, Steyn FJ, Wolvetang EJ and Ngo ST (2018) Neuronal Lipid Metabolism: Multiple Pathways LIPID SYNTHESIS, STRUCTURE AND TRANSPORT Driving Functional Outcomes in Health and Disease. In this section of the review, we provide an overview of the synthesis, structure, and Front. Mol. Neurosci. 11:10. transport of fatty acids, triacylglycerols (TAGs), phospholipids, sterol lipids and sphingolipids. doi: 10.3389/fnmol.2018.00010 The intracellular localization of the individual lipid classes is summarized in Figure 1. Frontiers in Molecular Neuroscience | www.frontiersin.org 1 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease An unsaturated fatty acid, however, contains a carbon chain in which double bonds have been introduced. Depending on the number of double bonds, the fatty acid can be classified as mono- or polyunsaturated. Polyunsaturated fatty acids (PUFAs) are of particular importance in the brain, where they serve as essential molecules for signaling and membrane structure (Bazinet and Layé, 2014). Another variation, although rare in humans, is the branching, or functionalization, of the fatty acid chain. For example, a saturated fatty acid has one or more methyl groups added to the carbon chain, significantly altering their physical properties, and, consequentially, ordering in cellular membranes (Ran-Ressler et al., 2008). The most common modification to fatty acids is microsomal fatty acid elongation, which predominantly takes place on the endoplasmic reticulum (ER). Malonyl CoA acts as the major substrate, and undergoes repeated cycles of condensation, reduction, dehydration and reduction reactions (Bressler and Wakil, 1962). Unlike the cytosolic process, which uses one enzyme complex only, microsomal chain elongation is coordinated by the action of four unique enzymes. The process is regulated primarily at the condensation step by the 3-keto acyl-CoA synthase enzyme (Moon and Horton, 2003). Seven forms of the enzyme have been characterized in humans, and each utilizes different input fatty acids, resulting in varying degrees of chain elongation (Leonard et al., 2004; Jakobsson et al., 2006). Chain elongation can also take place in the mitochondria, but this is far less common. The fatty acid FIGURE 1 | The structure of individual lipid classes affects their intracellular oxidation pathway that typically takes place in the mitochondria localization. Triacylglycerols (TAGs) consist of three fatty acid chains attached is reversed. Since this process is energetically unfavorable to a glycerol backbone. As TAG synthesis occurs in the endoplasmic reticulum (ER), a large proportion of TAGs are found within this compartment. A to reverse, the acyl-CoA dehydrogenase step is substituted significant proportion is also stored in specialized intracellular organelles for enoyl-CoA reductase. Such elongation also occurs in the known as lipid droplets. The subclasses of phospholipids are synthesized peroxisomes, in a similar fashion to the mitochondria (Alexson through a number of pathways. Phosphatidylcholine and phosphatidylinositol and Cannon, 1984; Wong et al., 2004; Demarquoy and Le are largely localized to the ER, while phosphatidylserine, phosphatidylglycerol Borgne, 2015). and phosphatidylethanolamine are mainly to localized to the mitochondria and its associated membranes. Sterol lipids, such as cholesterol, are highly Fatty acids can also be modified through desaturation, a concentrated at the plasma membrane. Sphingolipids are also largely process that introduces double bonds into the fatty acid chain. localized to the plasma membrane. This is particularly the case for Depending on the desaturase enzyme used, the position and sphingomyelin and glycosphingolipids. As major components of lipid rafts, number of double bonds differs. The most common desaturases sphingolipid concentration at the plasma membrane is kept high. are ∆9, ∆5 and ∆6, along with fatty acid desaturase 3 (FADS3; Nakamura and Nara, 2004). Desaturation is essential for diversifying fatty acid structure and function. Fatty Acids Fatty Acid Structure Fatty Acid Synthesis As the essential monomeric component of all lipid types, fatty Fatty acids are synthesized in the cytosol of lipogenic tissues. acids are an essential class of lipids. All fatty acids consist of While the brain can synthesize the majority of required saturated a carbon chain that terminates in a carboxylic acid functional and monosaturated fatty acids, it severely lacks the ability to group, and they are categorized into different subclasses based synthesize PUFAs (Moore, 2001). Fatty acid synthesis takes place on the length of the carbon chain. Fatty acids with 2–4 carbons over seven repetitions of a four-reaction cycle (Figure 2). Acetyl are classified as short-chain fatty acids, medium-chain fatty acids CoA, which is provided through the metabolism of glucose, is have 6–12 carbons, long-chain fatty acids have 14–18 carbons, first carboxylated to malonyl CoA via acetyl CoA carboxylase and very long-chain fatty acids have 18+ carbons. The length (ACC; Wakil et al., 1983). In humans, there are two major forms of the fatty acid introduces significant variation in function and of ACC: ACC1 and ACC2 (Abu-Elheiga et al., 1995, 1997; Ha subcellular localization (Agostoni and Bruzzese, 1992). et al., 1996). ACC1 is the minor form expressed in the cytosol of In fatty acids, the carbon chain is classified as either saturated human tissues, while ACC2 is the major form, and is expressed or unsaturated. A saturated fatty acid is defined by a carbon on the mitochondrial membrane (Castle et al., 2009). Expression chain in which all of the carbons are ‘‘saturated’’ with hydrogen is observed most highly in lipogenic tissues, such as adipose atoms. As such, only single bonds exist between the carbons. tissue and the liver, along with oxidative tissues, such as skeletal Frontiers in Molecular Neuroscience | www.frontiersin.org 2 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease The first reaction process is condensation. Acetyl ACP and malonyl ACP react to form acetoacetyl ACP, which is catalyzed by acyl-malonyl ACP condensing enzyme. Acetoacetyl ACP then undergoes reduction, to form D-3-hydroxybutyryl ACP, which is then dehydrated to form crotonyl ACP. Finally, crotonyl ACP is reduced to butyryl ACP, which marks the end of the first of seven elongation steps. As such, the first round of elongation ends with a 4-carbon chain product. Subsequent elongation cycles extend this carbon chain by two carbons each time, until a 16 carbon-acyl ACP (palmitoyl ACP) is formed, after seven repetitions. At this stage, a thioesterase enzyme catalyzes the formation of palmitic acid by terminating the carbon chain at the thioester bond. As a result, fatty acid synthesis is terminated with the formation of palmitic acid (Figure 2). Rather than relying solely on de novo synthesis and modification, the majority of fatty acids are provided from the diet. Short and medium-chain fatty acids are of particular importance as de novo synthesis is focused on the production of long and very long-chain fatty acids. Dietary fatty acids are mobilized directly into the bloodstream as triglycerides, and stored in adipocytes until required by the body. There are two fatty acids for which the body cannot synthesize sufficient quantities, and must rely on dietary sources. These are linoleic acid and α-linolenic acid, which are referred to as essential fatty acids (Ellis and Isbell, 1926). These dietary fatty acids are also susceptible to modification, further diversifying the fatty acid FIGURE 2 | Fatty acid synthesis is a cyclical process that utilizes a unique pool. The major products of linoleic acid and α-linolenic acid multifunctional enzyme complex. Glucose is metabolized to acetyl CoA through the standard glucose metabolic pathway. Acetyl CoA is then are arachidonic acid and docosahexaenoic acid—the major brain carboxylated by acetyl CoA carboxylase (ACC) to form malonyl CoA. This PUFAs, making a diet rich in essential fatty acids important for carboxylation step is the rate limiting process in fatty acid synthesis. Malonyl healthy brain function (Bazinet and Layé, 2014). CoA and unreacted acetyl CoA undergo transacylation to form malonyl ACP and acetyl ACP respectively. These intermediates enter fatty acid synthase (FAS), where four reactions take place; condensation, reduction, dehydration, Fatty Acid Transport and a second reduction. The four reactions add two carbons to the original Due to the functional importance of PUFAs in the brain, a carbon chain—in this case malonyl ACP. In humans, this cycle is typically significant portion of fatty acids must be imported from the repeated six more times, using the product from the end of the cycle as the blood. In such cases, fatty acids are metabolized from sources input molecules for the next cycle, producing palmitoyl ACP; a 16 carbon in adipose tissue or the bloodstream by lipoprotein lipase (LPL; product. A thioesterase enzyme then terminates the carbon chain at the thioester bond, forming palmitic acid. Goldberg et al., 2009). These ‘‘free fatty acids’’ then bind albumin in the blood as a carrier protein, and are transported throughout the circulatory system (Korn, 1955a,b). Upon reaching the CNS, muscle and the heart (Kreuz et al., 2009). ACC2 is also highly the fatty acids must pass the blood brain barrier. expressed in the brain and spinal cord (Castle et al., 2009). The Fatty acids are postulated to pass through the blood brain carboxylation of acetyl CoA to malonyl CoA is irreversible and barrier by two mechanisms. In the passive diffusion model, fatty the rate-limiting step in fatty acid synthesis (Ha and Kim, 1994). acids are hypothesized to dissociate from their albumin carriers After this carboxylation process, malonyl CoA acts as a and bind to the luminal membrane of the endothelial cell. Once carbon donor in the fatty acid synthesis process (Figure 2). The bound, the fatty acids diffuse across the membrane in a non- unreacted acetyl CoA and malonyl CoA intermediates undergo ATP-dependent manner and enter the cytosol. This process is transacylation to form acetyl ACP and malonyl ACP respectively. repeated for the transluminal membrane, allowing the fatty acids These serve as the starting point from which the four-reaction access to the brain extracellular space. From this point, they cycle repeats, and is facilitated by fatty acid synthase (FAS). FAS cross the plasma membrane of the neural cells, and reach their is a homodimer found within the cytoplasm that contains three target. This is referred to as the flip-flop method (Simard et al., domains and seven catalytic sites (Chirala et al., 2001). The first 2008). It is argued that this diffusion process is dependent on domain acts as the substrate entry and condensation unit, the the lipophilicity and size of the fatty acid (Kampf et al., 2006). It second domain acts as the reduction unit, and the third domain has been shown that short and medium-chain fatty acids easily acts as the fatty acid exit domain. As a result, the entirety of fatty cross the blood brain barrier due to their high permeability acid synthesis takes place within a single enzyme (Wakil, 1989; coefficients, while long-chain fatty acids are less permeable, Smith, 1994). and need to be in their non-ionized form for faster movement Frontiers in Molecular Neuroscience | www.frontiersin.org 3 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease (Kamp and Hamilton, 1992). Such differential diffusion speeds Therefore, fatty acids enter the brain through a number of have served as the major criticism of the diffusion model, different pathways; either via passive diffusion, or a host of where the significantly slower diffusion of the long-chain fatty transport-protein mediated pathways. As a result, the brain has acids may not be sufficient to supply the metabolic needs access to a range of fatty acids for metabolic purposes. of the CNS. Thus, faster ATP-dependent transporter protein- mediated mechanisms have been postulated. Involving four Triacylglycerol classes of transport proteins: Fatty acid transport protein (FATP), Compared to the other lipid classes, TAGs play a small role fatty acid translocase, Fatty acid binding proteins (FABPs) and in neuronal lipid metabolism. Nevertheless, they do act as the caveolae, these mechanisms can theoretically support the high storage form of lipid precursors. fatty acid metabolic rate of the brain (Mitchell and Hatch, 2011). FATP consists of six tissue specific isoforms. FATP-1 and Triacylglycerol Structure FATP-4 are the major isoforms found in the brain, and act on TAGs are composed of a glycerol backbone with three fatty acid the luminal membrane of the endothelial cells (Mitchell et al., chains. Variation in TAG structure arises from the fatty acid 2011). FATP-1 possesses specificity towards long chain fatty chains, which can vary in length, functionalization and degree acids (Watkins et al., 1998; Mitchell et al., 2011). ATP-dependent of saturation. The position at which these fatty acids are added transport of fatty acids has also been linked to long-chain to the glycerol backbone affects the physical and physiological acyl-CoA synthetase, marking it as an essential complex in properties of the TAG (Karupaiah and Sundram, 2007). multiple aspects of fatty acid metabolism. Fatty acid translocase/CD36 shows specificity for the long Triacylglycerol Synthesis and very long-chain fatty acids (Pepino et al., 2014). High The synthesis of TAG primarily occurs in adipose tissue and levels of expression are also observed in the brain, including the liver, but is also observed in skeletal muscle, kidney, on the luminal membrane of brain endothelial cells (Husemann lung, heart and the brain. TAG synthesis can occur via the et al., 2002). While the mechanism by which CD36 transports glycerol-3-phosphate, or the monoacylglycerol pathway in both fatty acids is not fully understood, studies suggest that the ER and the mitochondria (Weiss et al., 1960; Lehner CD36 promotes uptake via modifications to intracellular and Kuksis, 1996; Dircks and Sul, 1999). In the glycerol-3- metabolism (i.e., esterification), rather than directly increasing phosphate pathway, synthesis begins with glucose, which is the rate of fatty acid transport across the plasma membrane (Xu converted into glycerol-3-phosphate by a multi-step metabolic et al., 2013). reaction. Glycerol-3-phosphate, the rate-limiting step of TAG FABPs are made up of two subfamilies. Membrane-associated synthesis, is converted to lysophosphatidic acid by glycerol- FABPs are associated with the extracellular surface of the plasma 3-phosphate acyltransferase (GPAT). Lysophosphatidic acid is membrane, and bind all fatty acids with high affinity. The then converted into phosphatidic acid by 1-acylglycerol-3- mechanism of membrane-associated FABP-mediated transport phosphate acyltransferase (AGPAT), phosphatidic acid into is not wholly understood, but it is hypothesized that they 1,2-diacylglycerol by phosphatidic acid phosphatase (PAP), are not directly involved in transport, but rather act as and finally 1,2-diacylglycerol into TAG by diacylglycerol an intermediator between the free fatty acids and FATPs. acyltransferase (DGAT). The monoacylglycerol pathway is Cytosolic FABPs are more deeply understood. Multiple subtypes similar, but rather than initiating with glucose, this pathway of cytosolic FABPs show tissue specific expression (Glatz begins with monoacylglycerol, which is converted into 1,2- et al., 1997). Membrane transport of cytosolic FABP is diacylgycerol by monoacylglycerol acyltransferase (MGAT). The hypothesized to occur by one of two mechanisms. The first pathway then continues as for glycerol-3-phosphate. After is by aqueous phase diffusion through the membrane, where synthesis, TAGs are packaged into lipid droplets (Coleman and cytosolic FABP-fatty acid complexes passively diffuse through Lee, 2004). the plasma membrane, and the other is through collision transfer, TAG breakdown; a process also known as lipolysis, is essential where the FABP-fatty acid complex makes contact with the for the presentation of fatty acids to multiple tissue types. membrane to transfer the fatty acid (Storch and Thumser, Lipolysis principally occurs in the adipose tissue and is facilitated 2000). by a set of enzymes known as lipases (Ahmadian et al., 2007). Caveolae are intracellular invaginations of the plasma Lipolysis begins with the conversion of TAG into diacylglycerol membrane, for which three isoforms exist. Caveolin-1 has been by adipose triglyceride lipase (ATGL), releasing one fatty acid shown to have a major role in the transport of long-chain fatty molecule (Zimmermann et al., 2004). This reaction can also be acids (Pohl et al., 2002, 2005), and it is proposed that the caveolar catalyzed to some extent by hormone-sensitive lipase (HSL). domain is capable of budding off to form intracellular vesicles, Diacylglycerol is then further catabolized to monoacylglycerol by which then carries the fatty acids to subcellular organelles for HSL, releasing a second fatty acid molecule (Haemmerle et al., further processing (Stremmel et al., 2001). Fission of the vesicles 2002). Finally, monoacylglycerol is broken down to glycerol and with the plasma membrane transfers the lipid cargo from one the final fatty acid (Fredrikson et al., 1986). Lipolysis can also membrane to another. Caveolin is also postulated to indirectly occur in the bloodstream, as is required for the presentation of modulate fatty acid uptake by controling the localization of CD36 fatty acids to the blood brain barrier. In such cases, LPL catalyzes (Lobo et al., 2001; Ring et al., 2006). the reaction (Goldberg, 1996). Frontiers in Molecular Neuroscience | www.frontiersin.org 4 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease Triacylglycerol Transport 1958). The second is the phosphatidylethanolamine N- While TAG synthesis can take place in the brain, liver, heart, methyltransferase (PEMT) pathway, which synthesizes skeletal muscle and kidney, they are also transported to these phosphatidylcholines by the successive methylation of the regions through the bloodstream. In order to be transported, they phospholipid phosphatidylethanolamine (Shields et al., 2003; must be packaged into lipoproteins. Lipoproteins are composed Hörl et al., 2011). of a hydrophobic core of TAGs, cholesterol esters and fat soluble Phosphatidylethanolamine synthesis occurs through the vitamins, which are enveloped by a layer of phospholipids, free Kennedy pathway (Wright and McMaster, 2002; Figure 3). The cholesterol, and specialized apolipoproteins. There are multiple enzyme CTP:phosphoethanolamine cytidylyltransferase is the subtypes of lipoprotein, and they are categorized as high-density exception to this, and utilizes only phosphatidylethanolamine (HDL), low-density (LDL), intermediate-density (IDL) and very synthesis substrates (Vermeulen et al., 1993). While this form low-density lipoproteins (VLDL) based on their density. Another of synthesis takes place in the ER, phosphatidylethanolamine type of lipoprotein are the chylomicrons, and the size of these synthesis can also occur on the inner mitochondrial membrane, particles is controlled primarily by the number of TAG molecules through the phosphatidylserine decarboxylase pathway (Dennis that are incorporated (Jackson et al., 1976). and Kennedy, 1972). The majority of TAGs are transported by the chylomicrons Synthesis of phosphatidylserine is a process that relies on and VLDLs. Chylomicrons transport TAGs from dietary sources, phosphatidylcholines and phosphatidylethanolamines (Figure 3). while VLDLs deliver TAGs from endogenous sources (Dole and Phosphatidylserine is synthesized when the head group of Hamlin, 1962; Zilversmit, 1967; Jackson et al., 1976). A number either phosphatidylcholines or phosphatidylethanolamines is of other apolipoproteins are found on the lipoproteins surface, replaced by serine. Phosphatidylserine synthase 1 catalyzes and act as ligands and modulators of receptor and enzyme this reaction for phosphatidylcholines, while phosphatidylserine activity. Once lipoproteins reach their target tissue, they are synthase 2 catalyzes this reaction for phosphatidylethanolamines catabolized by LPL, releasing free fatty acids for cellular uptake, (Kuge et al., 1986). These reactions take place in the mitochondria- or can bind to lipoprotein receptors on the cell surface, leading to associated membranes of the ER (Stone and Vance, 2000). immediate incorporation (Goldberg, 1996). Phosphatidylinositol synthesis relies on phosphatidylinositol synthase to convert CDP-diacylglycerol—a modified version of Phospholipids diacylglycerol, directly to phosphatidylinositol (Antonsson, 1997; Phospholipids are a diverse class of lipids, with a wide Figure 3). This process occurs at the ER, but can also occur in range of roles in the human body. In particular, they hold ER-derived vesicles as well as the plasma membrane (Kim et al., great functional importance in the brain. Phospholipids 2011). Phosphatidylinositol is then heavily modified to produce a can be generally categorized into two groups; the number of essential signaling molecules, the importance of which glycerophospholipids, and the phosphosphingolipids. will be discussed later. Glycerophospholipids can be further categorized CDP-diacylglycerol is also essential for the synthesis of into phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and cardiolipin (Figure 3). On the inner phosphatidylserine, phosphatidylinositol, phosphatidylglycerol membrane of mitochondria, CDP-diacylglycerol is converted and cardiolipin (Li et al., 2015). Phosphosphingolipids will be to phosphatidylglycerol phosphate by phosphatidylglycerol considered in the context of sphingolipids later in this review. phosphate synthase, which is then further dephosphorylated to form phosphatidylglycerol (Kawasaki et al., 1999, 2001; Scherer Phospholipid Structure and Schmitz, 2011). Phosphatidylglycerol can then be further Phospholipids are composed of a glycerol backbone, with a modified by cardiolipin synthase, to form cardiolipin (Schlame hydrophobic fatty acid tail, a hydrophilic head group, and et al., 1993). a phosphate group. The type of head group identifies the phospholipid, with choline, ethanolamine, serine, inositol, or Phospholipid Transport glycerol groups capable of addition (Li et al., 2015). This While phospholipids are synthesized in their target cells at the hydrophobic/hydrophilic polarity defines the phospholipids as ER and mitochondria, intracellular transport is required amphipathic. for phospholipid utilization at appropriate membranes. Phospholipid transport is proposed to occur via soluble Phospholipid Synthesis transport proteins, vesicular transport, and close membrane Synthesis of all classes of phospholipids begins with two common contact between ‘‘acceptor’’ and ‘‘donor’’ membranes (Vance, precursors: phosphatidic acid and diacylglycerol (Figure 3). 2015). Phospholipid synthesis largely occurs in the ER in all major Localized within the cytosol, three forms of soluble tissue types (Vance et al., 1977; Coleman and Bell, 1978; Fagone transport proteins have been characterized. The first is the and Jackowski, 2009). In particular, the brain takes part in a phosphatidylcholine-specific transfer protein (PC-TP). Labeling substantial amount of phospholipid synthesis (Ross et al., 1997). studies show that PC-TP is essential for the movement of Phosphatidylcholines are synthesized through two major phosphatidylcholines from the ER to phosphatidylcholine- pathways (Figure 3). The first is the CDP-Choline/Kennedy deficient membranes, yet it does not appear to play a major role pathway, through which the majority of de novo synthesis in phosphatidylcholine-specific transport (Kanno et al., 2007). takes place (Kennedy and Weiss, 1956; Weiss et al., The second are phosphatidylinositol transfer proteins α Frontiers in Molecular Neuroscience | www.frontiersin.org 5 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease FIGURE 3 | All phospholipid subclasses are derived from phosphatidic acid via a variety of synthetic pathways. Phosphatidylcholine is synthesized via two pathways. The major pathway is the Kennedy pathway and involves the addition of CDP-choline to the phosphatidic acid derivative diacylglycerol. Phosphatidylcholine can also be synthesized through the action of phosphatidylethanolamine N-methyltransferase (PEMT), which converts phosphatidylethanolamine to phosphatidylcholine. Phosphatidylethanolamine is also synthesized via the Kennedy pathway, where the multifunctional enzymes catalyze the addition of CDP-ethanolamine to diacylglycerol. Phosphatidylethanolamine can also be synthesized by phosphatidylserine decarboxylase. Phosphatidylserine is synthesized from CDP-diacylglycerol, phosphatidylcholine, and phosphatidylethanolamine via phosphatidylserine synthase. Phosphatidylinositol is derived from the CDP-diacylglycerol by phosphatidylinositol synthase. Phosphatidylglycerol is synthesized from CDP-diacylglycerol via a multistep process involving phosphatidylglycerol phosphate synthase, and phosphatidylglycerol phosphate phosphatase. Cardiolipin can then be synthesized from phosphatidylglycerol via cardiolipin synthase. and β (PITPα and PITPβ). While their name suggests 1998). This suggests that if vesicular uptake of phospholipids phosphatidylinositol-specific action, their proteins are also at the plasma membrane is occurring, it is happening in capable of transporting phosphatidylcholine, although at a much non-protein associated vesicles. There also appears to be lower rate (Helmkamp et al., 1974). Robust characterization evidence that the transport of phospholipids from the plasma of the action of these proteins is still required, and as such, membrane back to the ER and mitochondria does occur, but most suggested functionality is only hypothesized based on this requires further validation (Sleight and Pagano, 1984, localization and structure. The final transport protein is the 1985). non-specific lipid transfer protein, which transports all classes The final proposed mechanism of phospholipid transport of lipids (Bloj and Zilversmit, 1977). Unlike the other transport involves close membrane association. In this model, close proteins, expression is also observed in the peroxisomes (Keller associations between membranes are formed by the action of et al., 1989; Mendis-Handagama et al., 1992). Knockdown of multiprotein tethering complexes. Indeed, electron microscopy this protein does not markedly affect phospholipid distribution, studies have shown such close associations between the ER and suggesting that it does not play a major role in intracellular mitochondria (Perkins et al., 1997), as well as the ER and plasma phospholipid transport (Seedorf et al., 1998). Thus, protein- membrane (Pichler et al., 2001). While phospholipid transport dependent intracellular transport does not appear to be a major between the ER and plasma membrane has yet to be observed contributor to phospholipid redistribution. in this manner, transport between the ER and mitochondria Vesicular phospholipid transport is not well characterized. has been well characterized. Membrane tether-associated The rationale behind this model is that vesicles that typically transport of phosphatidylcholines, phosphatidylethanolamines carry proteins to cellular membranes contain a bilayer of and phosphatidylserine has been observed in multiple cell types phospholipid containing molecules. When these vesicles fuse (Wu and Voelker, 2004; Horibata and Sugimoto, 2010; Tasseva with the membrane, it is expected that the phospholipids will et al., 2013). be incorporated into the new membrane (Vance, 2015). Studies of labeled phosphatidylcholines and phosphatidylethanolamines Sterol Lipids have shown that the uptake of these phospholipids occurs Sterol lipids are an essential class of lipids of particular on a different timescale compared to vesicular protein cargo importance to the brain. Sterol lipids are synthesized in many (Vance et al., 1991; Field et al., 1998; Huijbregts et al., forms, but the major form in all mammals is cholesterol. For Frontiers in Molecular Neuroscience | www.frontiersin.org 6 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease this review, all sterol lipids will be considered in the form of cholesterol. Sterol Lipid Structure Cholesterol is defined by its tetracyclic ring structure. A hydroxyl group is added to one end of this ring structure, which defines that portion as hydrophilic. The combination of the tetracyclic ring and hydroxyl group designates the sterol portion of the molecule (Tabas, 2002). A hydrocarbon chain is attached to the other end of the sterol. By nature, this hydrocarbon chain is hydrophobic, making cholesterol an amphipathic molecule. Sterol Lipid Synthesis All nucleated cells are capable of synthesizing cholesterol at the ER. The majority of cholesterol synthesis takes place in the liver, with significant quantities also being produced by the brain, intestines, and adrenal glands (Blom et al., 2011). Cholesterol synthesis acts through the mevalonate pathway, a complex series of reactions that utilize more than 20 steps (Langdon and Bloch, 1953; Bloch, 1965, 1992; Figure 4). Synthesis begins with acetyl CoA, which is converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), and then mevalonate. The rate-limiting and irreversible step of cholesterol synthesis is this conversion of HMG-CoA to mevalonate by HMG-CoA reductase. As such, it is heavily regulated at both FIGURE 4 | Cholesterol synthesis occurs through diverging pathways for different neuronal cell types. Cholesterol synthesis acts through the transcriptional and post-translational levels (Ye and DeBose- mevalonate pathway. Acetyl CoA is converted to 3-hydroxy-3-methylglutaryl Boyd, 2011). Mevalonate, via a six enzyme process, is converted CoA (HMG-CoA). HMG CoA is then converted to mevalonate via HMG-CoA to squalene. Lanosterol, the first cyclic intermediate in the reductase, and this is the rate limiting step in cholesterol synthesis. Via a synthesis pathway, is produced from squalene. At this stage, the complex series of reactions that involve multiple enzymes and reaction steps, synthesis pathways diverge into the Kandutsch-Russell or Bloch mevalonate is converted to squalene. Squalene is converted to lanosterol in an essential cyclization process. From here, the Kandutsch-Russel pathway, pathways—depending on the nature of the hydrocarbon chain, which is favored by neuronal cells, produces cholesterol from lanosterol, while before converging at the final product of cholesterol (Kandutsch the Bloch pathway synthesizes cholesterol that is favored by glial cells. After and Russell, 1960; Bloch, 1965). From this point, cholesterol cholesterol is synthesized, it can be further metabolized into vitamin D, steroid can be used to form bile acids, steroid hormones, or vitamin D hormones, or bile acids, or it may be incorporated into cellular membranes. (Figure 4). In the brain, the divergent pathways of cholesterol synthesis take precedence in different neuronal tissues. Neurons contain excess in the periphery, transport mechanisms are required to cholesterol variants that arise from the Kandutsch-Russell rebalance the distribution. In such cases, cholesterol is packaged pathway, while astrocytes favor Bloch pathway variants. In a into HDLs, which return it to the liver. In the CNS, lipoproteins similar vein, levels of the intermediate lanosterol are found are also used to transport cholesterol between multiple cell types to be much higher in neurons when compared to astrocytes. (Pitas et al., 1987; Orth and Bellosta, 2012). Cholesterol levels are also much higher in glial cells (Nieweg While the ER is the major site of cholesterol synthesis, et al., 2009). Together these findings have led to suggestions that the concentration of cholesterol at the ER is typically low neurons have lower capacity for cholesterol synthesis (Zhang and due to the rapid intracellular transport of cholesterol to Liu, 2015). appropriate membranes (Blom et al., 2011). A significant portion of cholesterol is transported to the plasma membrane Sterol Lipid Transport via non-vesicular mechanisms. This is proposed to occur Cholesterol transport occurs between and within cells. Dietary via the action of cytosolic carrier proteins, such as sterol sources of cholesterol are believed to account for as much carrier protein-2 (SCP-2; Puglielli et al., 1995; Gallegos et al., as 70% of total body cholesterol. As such, transport is an 2001), the oxysterol-binding protein-related proteins (ORPs; essential mechanism for appropriate cholesterol distribution. Zhao and Ridgway, 2017), and members of the steroidogenic Dietary cholesterol is transported from the gut to the liver, acute regulatory protein-related lipid transfer (START) family and then distributed throughout the body. For this to occur, (Gatta et al., 2015). Members of the START family have also intestinal enterocytes package cholesterol into chylomicrons, and been shown to be essential for the transport of cholesterol liver hepatocytes package cholesterol into VLDLs (Ikonen, 2008). into the mitochondria (Clark et al., 1994). A small portion Once in circulation, VLDLs are modified into LDLs, which of cholesterol is trafficked through the standard biosynthetic deliver cholesterol to the peripheral tissues. If cholesterol reaches Golgi complex secretory pathway, where it is presented to Frontiers in Molecular Neuroscience | www.frontiersin.org 7 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease the plasma membrane. Membrane trafficking via intracellular galactosyltransferase (CGT). Galactosylceramide is particularly LDL receptor-mediated uptake brings a portion of cholesterol enriched in the CNS. It is highly expressed in Schwann cells into the endosomal system, where it is recycled to the plasma and oligodendrocytes, and is also expressed, albeit at lower membrane and mitochondria (Sugii et al., 2003; Wojtanik levels, in spinal, cerebellar, and brainstem neurons (Schaeren- and Liscum, 2003). A final portion of free cholesterol is Wiemers et al., 1995). The remaining ceramide is transported esterified to form fatty acid sterol esters, which are packaged to the Golgi complex, where one of two enzymes catalyzes the into lipid droplets for storage (Robenek et al., 2006; Ploegh, synthesis of two complex sphingolipids. On the cytosolic side of 2007). As a result, intracellular cholesterol transport is in the Golgi, glucosylceramide synthase (GCS) converts ceramide a constant state of flux, with high levels of turnover at to glucosylceramide through the addition of a UDP-glucose the ER. group (Basu et al., 1968, 1973; Jeckel et al., 1992; Yamashita et al., 1999). On the luminal side of the Golgi, sphingomyelin Sphingolipids synthase (SMS) converts ceramide to sphingomyelin through the addition of a phosphocholine group (Huitema et al., Sphingolipid Structure 2004; Figure 5). Two forms of sphingomyelin synthase exist; Although various forms of sphingolipids exist, all are SMS1 and SMS2, and both are found on the trans-Golgi. SMS2, characterized by the inclusion of a sphingosine backbone. however, is also found on the plasma membrane, suggesting Sphingosine does not apply to a single structure, but is a broad that it may play some role in sphingomyelin metabolism term encompassing various modifications of a long chain base. at the plasma membrane (Ternes et al., 2009; Yeang et al., Long chain bases are either 2-amino-1,3dihydroxyalkanes/2- 2011). amino-1,3dihydroxyalkenes, with an alkyl chain between 14 and 22 carbons, and up to 2 double bonds (Chester, 1998). Significant Sphingolipid Transport branching and hydroxyl group additions can also occur. The transport of ceramide from the cytosolic to luminal surface Depending on the class of sphingolipid, a number of different of the ER is currently not understood. It is hypothesized to groups can be added to the sphingosine backbone. The simplest occur through spontaneous intrabilayer transport or via protein- sphingolipids are the ceramides, which consist of a sphingosine mediated transport (Hanada et al., 2003; Lopez-Montero et al., backbone linked to a fatty acid chain (Pinto et al., 2011). There is 2005; Hanada, 2010). Despite the suggestion of protein-mediated significant variation in the attached fatty acid, and mammalian transport, no transporter proteins have been implicated to sphingolipids typically contain saturated fatty acid chains of date. Transport from the luminal to cytosolic surface of the between 14 and 32 carbons (Merrill et al., 2005). Due to the Golgi complex, however, has been shown to be dependent inclusion of this fatty acid chain, sphingolipids are amphipathic. on non-vesicular FAPP2 transport protein (D’Angelo et al., Other classes of sphingolipids are introduced through the 2013). addition of various head groups to ceramide. Sphingomyelins Transport from the ER to the Golgi complex occurs via two result from the addition of phosphocholine to ceramide pathways (Figure 5). The first pathway involves the ceramide (Huitema et al., 2004). The addition of phosphocholine defines transfer protein (CERT), which transports ceramide in a non- these lipids as phospholipids, and they are therefore also referred vesicular, ATP-dependent fashion. It shows very high affinity to as phosphosphingolipids. Glycosphingolipids, also referred to for ceramide, and almost exclusively transports this ceramide for as cerebrosides, arise when one or more sugar residues are added sphingomyelin synthesis (Hanada et al., 2003). Consequentially, to ceramide. Glycosphingolipids of the brain typically have a it contains a number of highly regulated domains, which control galactose attached to the ceramide, while non-neuronal tissue this transport process (Levine and Munro, 1998; Loewen et al., favors glucose addition (Baumann and Pham-Dinh, 2001). 2003). The second pathway is less understood, and involves vesicular transport. Ceramide tracking studies have shown that Sphingolipid Synthesis vesicular transport machinery knockdown leads to defective Sphingolipid synthesis begins at the cytosolic leaflet of the ER, transport of ceramide derivatives (Fukasawa et al., 1999), but and progresses to several subcellular locations (Gault et al., direct evidence for vesicular trafficking is lacking. 2010; Figure 5). At the ER, this process begins with the condensation of palmitoyl CoA and serine to 3-ketosphinganine. 3-ketosphinganine is then reduced to dihydrosphingosine by THE FUNCTIONS OF LIPIDS IN NEURONS 3-ketosphinganine reductase. A family of (dihydro)ceramide synthases convert dihydrosphingosine to dihydroceramide. Lipids as Energy Substrates Ceramide synthase 1 is highly expressed in neurons in the The brain is an incredibly energy hungry organ. As such, it brain, while ceramide synthase 2, 5, and 6 are expressed requires a near constant source of metabolites to maintain at lower levels (Becker et al., 2008; Laviad et al., 2008). function. The general consensus is that this energy requirement is Finally, dihydroceramide desaturase converts dihydroceramide almost entirely satisfied by glucose metabolism. However, it has to ceramide. At this stage, ceramide is either used by the recently been shown that approximately 20% of the total energy cell, or transported elsewhere for further modification. A small requirement of the brain is met through the oxidation of fatty portion of ceramide is transported to the luminal leaflet of acids, and that this fatty acid oxidation may take place entirely in the ER, where galactosylceramide is generated by ceramide astrocytes (Ebert et al., 2003). Frontiers in Molecular Neuroscience | www.frontiersin.org 8 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease FIGURE 5 | Neuronal sphingolipid synthesis takes place across multiple cellular compartments. Sphingolipid synthesis begins at the cytosolic leaflet of the ER. Via a series of reactions, palmitoyl CoA and serine are converted to ceramide. A portion of this ceramide is transported to the luminal leaflet of the ER, where ceramide galactosyltransferase (CGT) converts the ceramide to galactosylceramide; an essential neuronal sphingolipid. Another portion of this ceramide is transported to the Golgi complex, where it is converted to either glucosylceramide on the cytosolic side of the Golgi via glucosylceramide synthase, or to sphingomyelin on the luminal side by sphingomyelin synthase. Transport of ceramide from the ER to the Golgi complex is facilitated by either ceramide transfer protein (CERT) or vesicular transport. Frontiers in Molecular Neuroscience | www.frontiersin.org 9 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease Pre-oxidation Steps the mitochondria for degradation. Similarly, the peroxisomal Fatty acid utilization for energy occurs through fatty acid β-oxidation pathway does not fully degrade fatty acids, only oxidation, which takes place in the mitochondrial matrix. chain-shorten them. Non-saturated and branched fatty acids In order to be oxidized, fatty acids are converted to fatty are metabolized via slightly different mechanisms. Due to the acyl-CoA by acyl-CoA synthases—with the subtype of variation in substrate structure, a number of ancillary enzymatic enzyme varying by fatty acid composition (Eaton et al., complexes are required for catabolism. As a whole, the enzymes 1996). Once this reaction takes place, the substrates are involved are multifunctional, with a wide range of substrate transported across multiple mitochondrial membranes to specificities (Hiltunen and Qin, 2000; Mannaerts et al., 2000; the mitochondrial matrix. This process is undertaken by Poirier et al., 2006). a set of proteins known as carnitine palmitoyltransferases A small amount of fatty acid oxidation, termed α-oxidation, (CPTs; McGarry and Brown, 1997). CPT1 on the outer also occurs in the peroxisomes. α-oxidation removes a single mitochondrial membrane converts fatty acyl-CoAs into fatty carbon from fatty acids that are incapable of typical β-oxidation. acylcarnitines, which are transported into the intramembrane For example, phytanic acid, which has a branched methyl group space through porins. CPT1 action is the rate-limiting step on the third carbon, is metabolized to pristanic acid through the of fatty acid oxidation, and is regulated by malonyl-CoA. α-oxidation pathway, allowing it to then be metabolized through This is particularly important, because at this stage, fatty the β-oxidation pathway (Singh et al., 1992, 1993; Pahan et al., acyl-CoAs can either be directed to oxidation for fuel in 1993; Jansen and Wanders, 2006). α-oxidation is particularly times of energy requirement, or to the formation of structural important in the brain, as build-up of phytanic acid causes glycerophospholipids in times of energy excess. Acylcarnatine neurological damage, as is seen in Refsum’s disease (Jansen et al., transferases then transport the fatty acylcarnitines across the 1997). inner mitochondrial membrane in exchange for free carnitine. CPT2 then reforms the fatty acyl-CoA. At this stage, the fatty Regulating Fatty Acid Oxidation acyl-CoAs are ready to enter the β-oxidation pathway (McGarry Although the rate-limiting step of fatty acid oxidation occurs et al., 1977, 1978; McGarry and Brown, 1997; Violante et al., through CPT1, significant regulation also occurs as the result 2010). of the energy sensing capacity of the cell. The ratio of [NADH] to [NAD+ ], which serves as a measure of cellular energetic Mitochondrial β-Oxidation status, regulates the activity of hydroxyacyl-CoA dehydrogenase Through a repeating sequence of four reactions that are (Eaton et al., 1998). Low ATP levels activate AMPK, leading to catalyzed by acyl-CoA dehydrogenase, enoyl-CoA hydratase, the inhibition of lipogenic enzymes (Hopkins et al., 2003). This hydroxyacyl-CoA dehydrogenase, and ketoacyl-CoA thiolase, pathway is also regulated via the action of specific transcription β-oxidation produces a considerable amount of energy from factors. Peroxisome proliferator-activated receptor α (PPARα), a single substrate (Eaton et al., 1996). Each cycle of reactions which is triggered in low energy states, increases the expression produces one molecule of FADH2 , one molecule of NADH, of a number of catabolic enzymes, such as the CPT family, and one molecule of acetyl CoA, and a fatty acyl derivative that acyl-CoA dehydrogenases (Leone et al., 1999). Low blood glucose is two carbons shorter than that in the previous step. This triggers the activation of CREB, activating a number of essential reaction cycle repeats until the entire carbon backbone has lipid catabolic enzymes (Herzig et al., 2003). been broken down. In cases of an odd numbered carbon chain, propionyl-CoA is produced as the final product (Mazumder Rationalizing Fatty Acid Oxidation et al., 1963; Jenkins et al., 2015). The FADH2 and NADH β-oxidation of palmitic acid produces seven FADH2 , seven incorporate their free electrons directly into the mitochondrial NADH and eight acetyl CoA molecules. These, in turn, produce electron transport chain for ATP generation, and the acetyl CoA 108 ATP molecules, giving a net gain of 106 ATP from a molecules enter the tricarboxylic acid cycle for further energy single molecule of palmitate (Reddy et al., 2014). By contrast, generation. This results in the generation of a significant amount the metabolism of glucose yields at most, 36 ATP per glucose of ATP (Lehninger, 1964; Balaban, 1990). Propionyl-CoA, molecule (Hinkle et al., 1991). There is a staggering discrepancy through a further three step catabolic pathway, is converted to between the amount of energy liberated from these molecules succinyl-CoA (Smith and Monty, 1959; Mazumder et al., 1961), and the frequency of use of these pathways. which acts as a substrate for gluconeogenesis via oxaloacetate The brain is a very fragile organ, with small changes in formation. environmental factors causing major disruptions. While accurate measurements of brain oxygen concentration are currently Peroxisomal β and α-Oxidation infeasible, the overall value can be described as low and Branched and very long-chain fatty acids are oxidized in nonuniform (Ndubuizu and LaManna, 2007). As anerobic the peroxisomes through the β-oxidation pathway (Lazarow glycolysis has low capacity for ATP generation, oxygen becomes and De Duve, 1976; Mannaerts and van Veldhoven, 1996). the limiting factor. In this regard, the oxidation of 1 mol of For long-chain fatty acids, the catabolism process occurs as palmitic acid requires 31 mol of oxygen, while oxidation of in the mitochondria, with a few minor differences. Since glucose requires 6 mol. Therefore, while palmitic acid may peroxisomes do not have a tricarboxylic acid pathway, the produce more ATP, it consumes significantly more oxygen to acetyl-CoA/propionyl-CoA metabolites are transferred back to do so. Frontiers in Molecular Neuroscience | www.frontiersin.org 10 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease The consumption of oxygen also liberates a number of found within the cell (van Meer et al., 2008). Lipid classes that harmful products, to which the brain is particularly vulnerable. take place in such functionality are the phospholipids, sterol Reactive oxygen species, particularly in the form of superoxide, lipids and sphingolipids. The common characteristic of these are generated in significant quantities from the β-oxidation lipids is that they are amphipathic, allowing them self-organize pathway. Within the mitochondria, electron leakage at various in aqueous environments to form lipid bilayers. Each lipid class steps in the electron transport chain produces superoxide however, also has a unique role in membrane structure and radicals. Due to the large number of electrons being channeled function. through this pathway via NADH/FADH2 , significant superoxide production occurs in this manner (Han et al., 2003; Turrens, Phospholipids and Cellular Membranes 2003; Murphy, 2009; Perevoshchikova et al., 2013). Although Phospholipids account for the majority of structural lipids in most superoxide radical formation occurs at the mitochondria, eukaryotic membranes. They form the major structural unit; peroxisomal β-oxidation also contributes. Free fatty acids have the phospholipid bilayer. They are heavily implicated in the also been shown to be capable of binding to electron transport plasma membrane, along with the Golgi, ER, endosomes and chain complexes, simultaneously decreasing the rate of oxidative mitochondrial membranes. Each of the subclasses play individual ATP generation, while increasing superoxide generation by roles, with varying structural characteristics imparting functional complexes I and III (Wojtczak and Schönfeld, 1993; Di Paola and variation. Lorusso, 2006). Phosphatidylcholine is the most abundant of the Therefore, while fatty acids are an ATP rich source of energy phospholipids in cell membranes. Phosphatidylcholine has for the brain, the inadvertent reliance on glucose, along with an almost perfect cylindrical molecular geometry. As a result, oxygen-centric functionality of the brain requires that glucose membranes composed of phosphatidylcholine do not feature must be used as the obligate substrate for energy production. any curvature (Thiam et al., 2013). While membranes consisting In cases of fasting or extreme exertion, fatty acid utilization can of phosphatidylcholine can be fluid or solid/gel-like, they are increase, but sustained fatty acid oxidation in this manner only typically fluid at room temperature. By altering the ratios serves to damage the brain. of phosphatidylcholine to other membrane phospholipids, the shape and permeability of the membrane can be altered. Ketone Bodies Modification of phosphatidylcholine to phosphatidic acid or During intense periods of fasting, fatty acid derivatives can lysophosphatidylcholine can also force the membrane into be used in another, less harmful manner for energy. This alternate geometries (van Meer et al., 2008). In the brain, process typically takes place in the liver, but has also been the majority of choline used for neurotransmission is stored observed in brain astrocytes. In conditions of low glucose, in the membrane as phosphatidylcholine (Blusztajn et al., fatty acid-derived acetyl-CoA is preferentially shuttled into 1987). As such, it serves as a vital reservoir for essential brain the ketogenesis pathway to form three major ketone bodies; function. acetoacetate, D-3-β-hydroxybutyrate and propanone (Garber Phosphatidylethanolamine is a minor component, and, et al., 1974; Balasse and Neef, 1975; McPherson and McEneny, for the most part, is found on the inner leaflet of the plasma 2012). After synthesis, these ketone bodies enter the bloodstream, membrane (Fadeel and Xue, 2009). Due to the relatively and are transported to peripheral targets, of which the major small head group, membranes with phosphatidylethanolamine target is the brain (Owen et al., 1967). Upon reaching their assume a conical geometry, with significant outwards curvature target, they undergo ketolysis. Reversion of acetoacetate back to (Thiam et al., 2013). Increases in phosphatidylethanolamine acetoacetyl-CoA (Serra et al., 1993) allows for the subsequent concentration also increase the fluidity of a membrane (Li et al., generation of acetyl-CoA, which then enters the tricarboxylic 2006). This is due to the nature of the fatty acyl chain, which is acid cycle to produce ATP (McPherson and McEneny, 2012). enriched in the PUFA arachidonic acid. In the brain, arachidonic The rationale behind ketogenesis is that it is primarily acid is an essential precursor to a number of important promoted by extrahepatic glucose levels, not necessarily the levels neuromodulatory molecules, such as the prostaglandins at the peripheral tissue (Robinson and Williamson, 1980). As and anandamides. The increased curvature and fluidity of such, the production of ketone bodies occurs at a site distant the membrane introduced by phosphatidylethanolamine is from the target. This is of particular importance to the brain, hypothesized to facilitate vesicular budding and membrane as acetyl-CoA formation, which is largely through β-oxidation, fusion, two essential neuronal processes (Glaser and Gross, 1995; produces a host of damaging oxidative by-products. By limiting Lohner, 1996). acetyl-CoA/ketone body synthesis to the liver, the brain decreases Phosphatidylserine is found largely on the inner leaflet oxidative stress, while increasing available energy. of the plasma membrane (Fadeel and Xue, 2009). As a negatively charged phospholipid, phosphatidylserine is thought Lipids as Cellular Structural Machinery to act as an electrostatic mediator for a number of proteins Perhaps the most essential role for lipids in the brain is as (Maksymiw et al., 1987; McLaughlin and Aderem, 1995). components of cellular structural machinery. This is particularly Phosphatidylserine may also act as a buffer for essential important in the brain due to the compartmentalization of the bioactive fatty acids. Docosahexaenoic acid, which accounts many signaling processes. While the major site of action of these for as much as 40% of all PUFAs in the brain, is essential lipids is the plasma membrane, they constitute all membranes for brain development and function, and is stored largely as Frontiers in Molecular Neuroscience | www.frontiersin.org 11 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease phosphatidylserine (Guo et al., 2007; van Meer et al., 2008). It interaction of the cholesterol molecules with phospholipids. is therefore hypothesized that phosphatidylserine in membranes This causes a condensation effect, whereby the lipid bilayer acts as a release/storage facility for docosahexaenoic acid. in these regions becomes tightly packed and ordered, creating Phosphatidylglycerol, in the context of eukaryotic a lipid ordered (lo ) phase (Ege et al., 2006; Ali et al., 2007). membranes, does not play a major role. A small component of In this phase, the membrane is still considered to be fluid, phosphatidylglycerol is observed in eukaryotic mitochondrial but the lipids within are in a much more ordered orientation. membranes (de Kroon et al., 1997; Morita and Terada, 2015). Such condensation also decreases membrane permeability Cardiolipin, as a metabolite of phosphatidylglycerol, is a in these regions (Bastiaanse et al., 1997). Interestingly, major constituent of mitochondrial membranes, accounting the association between phospholipids and cholesterol is for as much as 15% of all lipids. In the mitochondria, dependent on phospholipid subtype. Phosphatidylcholine is cardiolipin maintains the membrane potential of the inner the most highly associated, followed by phosphatidylserine and mitochondrial membrane, while also supporting proteins phosphatidylethanolamine. This is due to the nature of their involved in mitochondrial respiration (Jiang et al., 2000). sidechains, where cholesterol prefers to associate with saturated Phosphatidylinositol does not play a major role in membrane fatty acyl chains, to promote closer packing (Ohvo-Rekilä et al., structure. It does, however, play major roles in membrane-bound 2002). signaling processes and vesicular activity, which will be discussed Depending on both the concentration of cholesterol, as well as in the following section. the temperature of the membrane, cholesterol can have differing effects. At low concentrations cholesterol has a minor effect on Sphingolipids and Cellular Membranes membrane composition, and most phospholipid membranes are The structural role of sphingolipids in membranes facilitates in a lipid disordered state. As cholesterol concentration increases, their role in signaling processes. The hydrophilic head groups the membrane becomes more ordered, until crystallization contain a number of hydroxyl groups, which allow for extensive begins to occur (Bach and Wachtel, 2003). At high temperatures, hydrogen bonding between individual head groups (Pascher, the tight packing of fatty acyl chains with cholesterol decreases 1976; Boggs, 1987). This creates a flexible surface membrane that the fluidity of the membrane, while at low temperatures, is largely impermeable. The fatty acyl groups that are associated the presence of cholesterol hinders the tight packing that is with sphingolipids allow for thicker and more closely packed required for highly ordered membranes (Khan et al., 2013). membranes. As a result, sphingolipids act as determinants Thus, cholesterol acts as a buffer for temperature-dependent of membrane fluidity and permeability (Pascher, 1976). A membrane fluidity, limiting the extremes typically observed in concentration gradient of sphingolipids is observed in cellular a cholesterol-free membrane. Despite these biophysical effects of membranes. The ER has a low concentration, the Golgi has cholesterol, the exact mechanism behind them is still unknown. an intermediate concentration, and the plasma membrane and Cholesterol and sphingolipids also show close associations in endosomes have a high concentration. This gradient is in place the brain through lipid rafts. Along with the phase separation to align with cellular function. The ER has a low concentration observed as the result of sphingolipid association, it is also since a more fluid membrane allows for easier protein insertion understood to occur as the result of close associations between and folding, whereas a high sphingolipid concentration in the sphingolipids and cholesterol. A number of calorimetric and plasma membrane and endosomes creates thicker and less cholesterol partitioning experiments have shown that the affinity permeable barriers to outside molecules (van Meer et al., of cholesterol for sphingolipids is above that of phospholipids 2008). due to the amide linkage found in sphingolipids. Therefore, such Another structural component that sphingolipids take part close associations drive further phase separation between the in are lipid rafts. These lipid rafts are the result of the sphingolipids and phospholipids, promoting the formation of strong intermolecular forces between individual sphingolipid these raft structures. Furthermore, the liquid ordered state, as molecules, driving a phase separation of the sphingolipids from facilitated by cholesterol, is hypothesized to be the phase required the phospholipid-rich outer membrane (Brown and London, for lipid raft formation (Silvius, 2003). 2000; Bacia et al., 2005). Present on membranes with high concentration of sphingolipids and cholesterol, lipid rafts act as major anchoring sites for proteins. Proteins that integrate with Lipids as Bioactive Molecules these rafts have been implicated in a host of processes, ranging As bioactive molecules, lipids take part in a wide range of from endocytic pathway sorting to antigen-responsive signaling cellular signaling processes. Here, signaling processes will only (Posse de Chaves and Sipione, 2010). be reviewed in the context of the CNS. Fatty acids and their derivatives have been well characterized as drivers of Sterol Lipids and Cellular Membranes intracellular signaling processes (Graber et al., 1994). One class Cholesterol plays a major role in determining cellular membrane that show particularly well-defined roles are the PUFAs. As flexibility and permeability. This is achieved through complex previously mentioned, the brain is enriched in two major PUFAs; interactions of cholesterol molecules with the phospholipid arachidonic acid and docosahexaenoic acid. Consequentially, bilayer. The structurally rigid planar ring structure—the PUFAs have been implicated in neuronal signaling processes sterol group, is the major facilitator of this (de Meyer and controling neurogenesis, brain vesicular activity, central glucose Smit, 2009). The polar nature of this group causes close homeostasis, mood and cognition (Bazinet and Layé, 2014). Frontiers in Molecular Neuroscience | www.frontiersin.org 12 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease Unmodified PUFAs primarily act upon fatty acid-activated Sphingolipids also play a major signaling role in the brain. receptors. The most well studied family of receptors are the The brain contains a high concentration of gangliosides, PPARs. In the brain, PPARδ and PPARβ are involved in the which are synthesized through the addition of sialic acid to regulation of fatty acid metabolism and inflammatory responses glycosphingolipid monomers (Yu et al., 2011). In neuronal (Tyagi et al., 2011). PUFAs also downregulate SREBP1 activity, membranes, gangliosides make up as much as 12% of total which is involved in de novo lipogenesis (Infantino et al., 2007). lipid content (Posse de Chaves and Sipione, 2010). Throughout This effect is further enhanced by the action of PUFAs on liver development, the composition of brain gangliosides switches X and retinoid X receptors (LXRs and RXRs), where PUFA from predominantly simple gangliosides (GM3) to complex binding inhibits SREBP1 activation via LXR/RXR (Yoshikawa gangliosides (GM1a). Such changes in the expression patterns of et al., 2002). gangliosides suggest a role in brain development (Yu et al., 1988; PUFAs are also involved in more distinct signaling pathways. Ngamukote et al., 2007). Taken together, gangliosides have been Endocannabinoids are fatty acid derivatives, with the major shown to have major roles in membrane protein modulation, forms in the brain being the arachidonic acid derivatives cell-cell adhesion, axonal growth, synaptic transmission, neural anandamide, and 2-arachidonoylglycerol. These bind to development and differentiation and receptor regulation (Yu cannabinoid receptor type 1 and 2 on both neurons and et al., 2011). glia (Matsuda et al., 1990; Tsou et al., 1998; Howlett and In many cases, a combination of lipids facilitates signaling Mukhopadhyay, 2000). Acting as retrograde messengers at events. This is particularly the case for lipid rafts. The close type 1 receptors, they supress neurotransmitter release (Kim association of phospholipids, cholesterol and sphingolipids leads and Thayer, 2000). At excitatory and inhibitory synapses to the formation of lipid rafts (Simons and Sampaio, 2011). Lipid this mediates short-term synaptic plasticity and long term rafts serve as major organizing centers for proteins and signaling depression (Gerdeman et al., 2002; Chevaleyre and Castillo, molecules, acting as essential cellular signaling components 2003; Kano et al., 2009). While this occurs largely on neurons, (Allen et al., 2007). In the brain, lipid rafts have been implicated endocannabinoids have been shown to mediate these effects in ionotropic receptor localization, binding and trafficking, through glial cell receptors (Hong et al., 2003). neurotransmitter transport, cytoskeletal rearrangement through PUFAs also play a major role in inflammatory signaling tubulin and actin remodeling, exocytosis, organization of pathways. Interestingly, the structure of the PUFA can G-protein coupled receptor machinery assembly for downstream significantly alter inflammatory response, where omega-3 signaling, cell surface receptor clustering, metabolism, neuronal fatty acids have an anti-inflammatory effect in the brain (Calder, growth and development, and redox signaling (Allen et al., 2010), and omega-6 fatty acids have a pro-inflammatory 2007; Benarroch, 2007; Jin et al., 2011). Thus, lipid rafts are effect (Patterson et al., 2012). Consequentially, expression the central organizing space for all major classes of neuronal of docosahexaenoic acid and its intermediates have been processes. shown to have a potent anti-inflammatory effect by lowering In summary, neuronal lipid signaling occurs through a range levels of pro-inflammatory cytokines in the brain following of processes, some driven by individual lipid classes, while others LPS administration (Delpech et al., 2015). Studies have also require more complex associations. The result is an incredibly shown that diets rich in docosahexaenoic acid lower the risk of intricate system that has multiple layers of redundancy, ensuring neuroinflammatory diseases (Minogue et al., 2007). Arachidonic tightly controlled processes. acid intermediates, however, are potent neuroinflammatory enhancers. Major metabolites of arachidonic acid are the prostaglandins, which have been heavily implicated in NEURONAL LIPID METABOLISM IN inflammatory responses throughout the body. Their expression AMYOTROPHIC LATERAL SCLEROSIS is particularly high under pathogenic neuroinflammatory conditions, suggesting a critical role in brain pro-inflammatory Amyotrophic Lateral Sclerosis responses (Ricciotti and FitzGerald, 2011; Lima et al., ALS is a progressive neurodegenerative disorder that is 2012). characterized by the selective degeneration of upper motor Phosphorylated forms of phosphatidylinositol activate neurons in the motor cortex and lower motor neurons in the phospholipase C, creating inositol triphosphate (IP3 ) and brainstem and spinal cord. The progressive degeneration of diacylglycerol (Berridge and Irvine, 1984; Vanhaesebroeck these motor neurons leads to paralysis, and eventual death et al., 2001). IP3 is transported rapidly to the cytosol where within 2–5 years from diagnosis (Kiernan et al., 2011). Despite it promotes calcium release (Berridge, 2009). In this way, the breadth of research on ALS, its etiology is still not well phosphatidylinositol signaling in the brain has been linked to understood. A growing number of in vitro and in vivo studies inter-neuronal communication through vesicular-mediated have begun to investigate metabolism as a means of explaining action of muscarinic and serotonergic receptors. Diacylglycerol the neuropathology observed in ALS. While a number of can either be phosphorylated to give the phospholipid precursor metabolic hallmarks have been observed in ALS patients (Reyes phosphatidic acid (Rodriguez de Turco et al., 2001), or et al., 1984; Desport et al., 2005; Dupuis et al., 2008; Pradat et al., hydrolyzed to arachidonic acid precursors (Bell et al., 1979). 2010; Jésus et al., 2018), interesting alterations in lipid handling In this way, diacylglycerol can give rise to a host of signaling mechanisms have also been noted to occur (Dupuis et al., 2008; processes, through two diverging pathways. Dorst et al., 2011). Frontiers in Molecular Neuroscience | www.frontiersin.org 13 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease A major site of interest for lipid studies in ALS is skeletal muscle. Many studies have suggested that skeletal muscle is a major source of dysregulated lipid metabolism. Indeed, a defined switch from glucose-based to lipid-based metabolism is an early pathological event in ALS muscle (Palamiuc et al., 2015). Furthermore, significant alterations in glycosphingolipid metabolism in the muscle of ALS mice impacts muscle innervation and motor recovery (Henriques et al., 2015b, 2017). Thus, dysregulation in lipid metabolism in skeletal muscle have been linked to pathological outcomes. CNS-Specific Alterations in Lipid Metabolism in ALS Having reviewed the multiple functions of lipids individually, we will now frame the dysfunctions caused by abnormal lipid metabolism in ALS in this way. Dysfunctions in Lipids as an Energy Substrate A growing focal point in ALS research is the role of lipids as an energy substrate. Given consistent observations of altered lipid metabolism in skeletal muscle, research has begun to consider neuronal lipid energy use in ALS. Such research, however, is still in its infancy. Perhaps the most compelling evidence towards a pathological role for lipid metabolism in ALS neurons is through CNS-specific oxidative stress, in which a range of lipid- derived oxidative pathway intermediates have been observed at FIGURE 6 | Fatty acid oxidation is a major contributor to reactive oxygen heightened levels in the CNS (Tohgi et al., 1999; Bogdanov et al., species production, which is increased in amyotrophic lateral sclerosis (ALS). 2000; Simpson et al., 2004; Mitsumoto et al., 2008). With the Although fatty acids are not the obligate substrate for energy production in the discovery of the superoxide dismutase-1 (SOD1) mutant in ALS, cell, β-oxidation of fatty acids generates a substantial amount of reactive oxygen species as a by-product. In turn, these promote a number of harmful researchers were quick to pin the cause of oxidative stress on oxidative effects including lipid peroxidation, protein oxidation, DNA damage, this mutation (Rosen et al., 1993; Wiedau-Pazos et al., 1996; and apoptosis. As neurons are not effectively equipped to deal with oxidative Andrus et al., 1998). Further studies have determined that while stress, these harmful effects are multiplied, contributing to neurodegeneration. the SOD1 mutation may contribute to oxidative stress, it is not the major cause. This is supported by the presence of oxidative stress in non-SOD1 ALS (Duan et al., 2010; Braun et al., 2011; (Fergani et al., 2007; Dodge et al., 2013). Similarly, elevated Iguchi et al., 2012; Kiskinis et al., 2014; Carri et al., 2015; Hirano levels of ketone bodies have been observed in ALS patient et al., 2015; Zhan et al., 2015). cerebrospinal fluid (Blasco et al., 2010; Kumar et al., 2010). In light of this, researchers have considered energetic With the suggestion of increased peripheral lipid availability, substrate metabolism as a source of oxidative stress. In the it is plausible that metabolic utilization of these lipids would brain, neuronal metabolism is largely aerobic, and involves increase. Consequentially, studies of mouse models of ALS, as glucose/lactate, while glial cell metabolism is anaerobic (Itoh well as in ALS patients, show that markers of oxidative stress et al., 2003; Herrero-Mendez et al., 2009). During normal and lipid peroxidation are significantly elevated in brain and neuronal activity, oxidative stress is kept relatively low (Almeida spinal cord tissue, via lipid-centric pathways (Simpson et al., et al., 2004). In ALS, however, an increased demand for energy 2004; Miana-Mena et al., 2011). Hence, an increased focus is placed on the motor neurons. Despite this, brain and spinal on lipid metabolites as a fuel source would inevitably lead cord glucose use (Hatazawa et al., 1988; Browne et al., 2006), as to increased oxidative stress, which would have number of well as the concentration of tricarboxylic acid cycle intermediates deleterious outcomes (Figure 6). are significantly decreased (Niessen et al., 2007). Similarly, In ALS patient muscle, peroxisome proliferator-activated reduced lactate transport and metabolism (Lee et al., 2012), receptor gamma coactivator 1-α (PGC-1α), a master damaged neuronal mitochondria (Magrané et al., 2014), and regulator of normal mitochondrial function and biogenesis, is mitochondrial electron transport chain dysfunction (Crugnola downregulated, leading to modifications in fatty acid signaling, et al., 2010; Shi et al., 2010; Smith et al., 2017) highlight that in and increased β-oxidation (Barroso et al., 2011; Thau et al., ALS, the brain has a significantly decreased ability to metabolize 2012). In mouse models of ALS, downregulation of PGC-1α has glucose for fuel. It is therefore hypothesized that alternate been shown to hasten disease progression (Eschbach et al., 2013), substrates are metabolized to meet the energy requirements of while upregulation of PGC-1α has been shown to maintain the brain. Indeed, in mouse models of ALS, lipid catabolism healthy levels of mitochondrial biogenesis in muscle, as well and clearance to peripheral tissues is significantly increased as improve muscle function (Da Cruz et al., 2012). Survival, Frontiers in Molecular Neuroscience | www.frontiersin.org 14 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease FIGURE 7 | Dysregulated lipid metabolism exerts a multifaceted effect on neurons in ALS. Dysregulation of neuronal lipid metabolism in ALS impacts energy use, structural integrity and signaling processes. Increased use of lipid as an energy substrate leads to increased oxidative stress. This exacerbates inflammation, mitochondrial dysfunction, metabolic dysfunction and excitotoxicity. Altered lipid metabolism also disrupts intracellular lipids leading to cytoskeletal defects and the denervation of neuromuscular junctions. Finally, changes in lipid metabolism impacts the composition of lipid rafts. This disrupts signaling processes that are crucial in regulating neurotransmitter synthesis and release, cytoskeletal integrity and intracellular transport. however, is not extended. Together, these findings highlight strong case can be made for the role of PGC-1α in maintaining the essential link between fatty acid oxidation and disease, CNS-driven fatty acid metabolism. In a similar fashion, the while suggesting that muscle may not be the primary target. stearoyl-CoA desaturase 1 (SCD-1) gene has been implicated in Application of these findings to a neuronal model, therefore, ALS. SCD-1 is a key enzyme in fatty acid metabolism regulation, would be expected to have more drastic effects, given the poorer and directly alters the levels of β-oxidation that occur in the oxidative defense capabilities of the CNS. Interestingly, when mitochondria (Ntambi, 1999; Ntambi et al., 2002). In mouse PGC-1α is upregulated in the CNS, mitochondrial function is models of ALS, as well as ALS patient muscle samples, SCD-1 has not only improved centrally, but motor function and survival been shown to be downregulated (Pradat et al., 2012; Hussain are also drastically improved (Zhao et al., 2011). Therefore, a et al., 2013). While downregulation of SCD-1 may explain Frontiers in Molecular Neuroscience | www.frontiersin.org 15 January 2018 | Volume 11 | Article 10 Tracey et al. Neuronal Lipid Metabolism in Health and Disease increased expression of β-oxidation enzymes, increased energy downregulation of pathway intermed