REVIEW ARTICLE

Adapting Brain Metabolism to Myelination and Long-Range Signal Transduction Johannes Hirrlinger1,2 and Klaus-Armin Nave1 In the mammalian brain, the subcortical white matter comprises long-range axonal projections and their associated glial cells. Here, astrocytes and oligodendrocytes serve specific functions during development and throughout adult life, when they meet the metabolic needs of long fiber tracts. Within a short period of time, oligodendrocytes generate large amount of lipids, such as cholesterol, and membrane proteins for building the myelin sheaths. After myelination has been completed, a remaining function of glial metabolism is the energetic support of axonal transport and impulse propagation. Astrocytes can support axonal energy metabolism under low glucose conditions by the degradation of stored glycogen. Recently it has been recognized that the ability of glycolytic oligodendrocytes to deliver pyruvate and lactate is critical for axonal functions in vivo. In this review, we discuss the specific demands of oligodendrocytes during myelination and potential routes of metabolites between glial cells and myelinated axons. As examples, four specific metabolites are highlighted (cholesterol, glycogen, lactate, and N-acetyl-aspartate) that contribute to the specific functions of white matter glia. Regulatory processes are discussed that could be involved in coordinating metabolic adaptations and in providing feedback information about metabolic states. C GLIA 2014;62:1749–1761 V

Key words: myelin, oligodendrocyte, axon, astrocytes, energy metabolism, cholesterol

Introduction

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any cell types develop fine processes, but the outgrowth of axons is a unique feature of neurons in the nervous system. Axonal projections allow the propagation of action potentials from neuronal somata to distant target cells millimeters or even meters away. Thus, neuronal axons extend further than any other known cellular process and more than 99% of the cellular mass of a projection neuron can be axonal. In the central nervous system, most of these long axons run within white matter tracts, characterized by a unique cellular composition that includes long axons, myelinating oligodendrocytes, astrocytes, NG2 cells, and microglial cells. Even under steady state conditions the energy consumption of myelinated axons devoted to axonal repolarization, axonal transport, and maintenance of glial cells, is significant (Harris and Attwell, 2012). Capillaries of the vasculature in the subcortical white matter, being the major source of energy substrates, have about half the spatial density as in cortical grey matter (Heinzer et al., 2008). Because

of these spatial dimensions the development and function of white matter tracts in the mammalian CNS faces unique challenges of metabolism. The issues of axon length and metabolism are coupled in many ways. Axonal glycolytic enzymes are, as far as we know, synthesized in neuronal somata. While some enzymes like GAPDH may also move fast bound to vesicles (Zala et al., 2013), most soluble enzymes are found in the slow axonal transport fraction SCb, moving a few millimeters per day (Brady and Lasek, 1981; Oblinger et al., 1988; Yuan et al., 1999). Thus, for meter long axons in the spinal cord (the longest ones extend into the peripheral nervous system) it might take years for enzymes to reach distal parts of the axon, likely exceeding their functional stability at 37 C (Nave, 2010b). Fast transport of mRNA in ribonuclein particles (RNPs) and local protein translation is a theoretical alternative, which has been studied in short axons and in brain development (Jung et al., 2012). However, it is less clear whether the entire machinery of protein translation can

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22737 Published online August 6, 2014 in Wiley Online Library (wileyonlinelibrary.com). Received Oct 21, 2013, Accepted for publication July 23, 2014. Address correspondence to Johannes Hirrlinger, Department of Neurogenetics, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 G€ ottingen, Germany or Carl-Ludwig-Institute for Physiology, Liebigstr. 27, D-04103 Leipzig, Germany. E-mail: [email protected] or [email protected] ottingen, Germany; 2Carl-Ludwig-Institute for Physiology, Faculty of From the 1Department of Neurogenetics, Max-Planck-Institute for Experimental Medicine, G€ Medicine, University of Leipzig, Leipzig, Germany.

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FIGURE 1: Metabolic cooperation of axons, oligodendrocytes, and astrocytes in developing CNS white matter. During myelination metabolism is directed towards formation of myelin. Glucose, lactate, and N-acetyl-aspartate (NAA) are used to generate acetyl-CoA for myelin lipid synthesis including cholesterol. While NAA is derived from neurons via a not yet fully resolved pathway, lactate can be delivered from astrocytes either directly via gap junctions (GJ) or via release and re-uptake via monocarboxylate transporters (MCT). CoA: coenzyme A; GLUT: glucose transporter; Glc: glucose; Lac: lactate; Pyr: pyruvate. Adapted with permission from F€ unfschilling et al., Nature, 2012, 485, 517–521, C Nature Publishing Group. V

maintain itself in a distant axonal compartment for years. Thus, it is plausible that metabolites for maintaining axonal functions are also locally derived, utilizing enzymes localized in axon-associated glial cells. In fact, metabolic support may be a key function of local glial cells that are a feature of all axons, also in invertebrate species and in the absence of a myelin sheath. The picture is further complicated by the arrangement of different cell types within white matter tracts. Oligodendrocytes directly contact multiple axons providing electrical insulation for saltatory impulse propagation by their myelin sheaths, but also forming a physical shield around the axon. This may limit the rapid access of metabolites from the extracellular milieu to the axonal cytoplasm, at least in myelinated fibers with long internodes (Nave, 2010b). Fibrous astrocytes of the white matter contact capillaries with their end feet (Butt and Ransom, 1989; Butt et al., 1994a; Suzuki and Raisman, 1992) and become part of the blood brain barrier (BBB), being the first in line to take up metabolites from blood (overview: Daneman, 2012). Fibrous astrocytes also directly contact axons at the nodes of Ranvier (Black and Waxman, 1998; Butt et al., 1994b; Raine, 1984). However, the percentage of white matter astrocytes residing in such a strategic position and the fraction of all nodes along a myelinated axon in contact to astroglial processes is unclear. Astrocytes are coupled by gap junctions to each other (notably by connexins Cx43 and Cx30; for details: Giaume et al., 2010) and by Cx47 and Cx32 to oligodendrocytes and non-compacted myelin compartments. This continuum of glial cytosolic compartments extends even through the myelin 1750

sheath. Non-compacted regions are routes for vesicular trafficking and metabolite diffusion that may be important for myelin biogenesis and for metabolic coupling throughout adult life. Oligodendroglial cytoplasm extends into myelinating processes (shafts) continuing within each internode as a nanometer-wide system of “myelinic channels” (outer collar/ mesaxon, paranodal loops, inner lip/mesaxon) that finally faces the thin periaxonal space underneath the myelin sheath. This architecture of the myelinated axon and its associated glial cells is the morphological underpinning for the metabolic interactions discussed in this review. Building and maintaining CNS white matter is metabolically challenging (Fig. 1). In early development, axons grow through the (prospective) white matter region prior to arrival of most glial cells and when projection axons are relatively “young” (days/weeks) and “short” (mm/cm). Only after neuronal connections are established, precursors of astrocytes and oligodendrocytes expand in numbers and differentiation of oligodendrocyte precursors (OPC/NG2 cells) leads to myelination. At this stage oligodendrocytes produce large amounts of lipids and proteins within a few days, synthesizing the equivalent of their own mass in less than 8 hr (Czopka et al., 2013; Pfeiffer et al., 1993; Simons and Trajkovic, 2006). After completion of myelination, a second metabolic challenge is maintaining these structures, including the myelin sheath, which can be considered an external oligodendroglial organell. While myelin proteins, specifically of the compacted membranes, have an unusual long half-life (Toyama et al., 2013), the complex architecture makes membrane turnover and delivery within the mature myelin sheath difficult and slow. A major function of metabolism in adult white matter concerns the energetics of action potential propagation and ATP-driven axonal transport processes, i.e. the necessary energy supply (Fig. 2). This developmental change of metabolic tasks is most obvious for oligodendrocytes and the metabolic changes are reminiscent of those in glycolytic tumor cells (“Warburg effect”, i.e. cells obtain energy mainly by glycolysis despite the presence of oxygen). It has been estimated that import of glucose at the node of Ranvier might suffice to maintain mitochondrial ATP generation in axons without additional delivery of glycolysis products by glial cells (Harris and Attwell, 2012). However, nodal glucose uptake might not be sufficient in fibers with longer internodes, larger calibers, or higher frequency of action potentials than those of optic nerve, on which the parameters for the calculations were based. Nevertheless, the node of Ranvier is strongly involved in the energetics of action potential propagation as the sodium gradient is dissipated mainly there (Waxman and Ritchie, 1993). Furthermore, white matter metabolism includes interactions of pathways in different cell types that support each other. When glucose levels are low, astrocytes Volume 62, No. 11

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FIGURE 2: Glial support of axonal energy metabolism. After completion of myelination, metabolism is adjusted to support axonal energy needs, required for maintaining membrane potential and axonal transport rates. Energy-rich substrates (lactate or pyruvate) are released from glial cells and reach the axonal compartment. Lactate derived from astroglial glycogen via glycolysis is transferred directly to the axon at the node of Ranvier or after transfer to oligodendrocytes via oligodendroglial MCT1 directed towards the axon. The function of NAA released from the axon and hydrolyzed by oligodendrocytes is not yet clear. Paranodal gap junctions connecting the different layers of myelin membranes are also illustrated. Abbreviations as in Fig. 1; Asp: aspartate. Adapted with permission from F€ unfschilling et al., Nature, C Nature Publishing Group. 2012, 485, 517–521, V

help maintaining axonal firing rates by providing lactate, derived from stored glycogen (Brown and Ransom, 2007). Oligodendrocytes contribute to energy supply of myelinated axons by delivering pyruvate/lactate and this support is essential for normal axon function in vivo (Funfschilling et al., 2012; Lee et al., 2012; Nave, 2010b). The picture emerges that all cellular compartments in the white matter—axons, oligodendrocytes and astrocytes – are metabolic cooperation partners (Amaral et al., 2013). Since this topic easily fills large volumes, we will focus here on selected aspects: (i) possible transcellular routes of metabolites to reach their final destinations; (ii) selected metabolic pathways contributing to white matter development and adult physiology; and (iii) possible mechanisms regulating these metabolic interactions.

“Structural Metabolism”: Building Myelin The formation of a white matter tract is a major developmental task accomplished in steps: (1) neurons grow axons that run in fiber bundles and leave the prospective white matter tract only at appropriate positions to connect to target cells; (2) oligodendrocyte precursor cells migrate into these fiber bundles and proliferate, before differentiating into oligodendrocytes; (3) cellular processes of mature oligodendrocytes target axonal segments and wrap multilayered myelin membranes; (4) secondary growth results in formation of compacted myelin sheaths and increased axonal diameters, associated with formation of functional nodes of Ranvier; (5) November 2014

astrocytes contact axons (at the nodes of Ranvier), oligodendrocytes, and myelin compartments, as well as endothelial cells at capillaries, thereby establishing transport routes between these cells. The major biosynthetic effort is made by oligodendrocytes. During myelination, one oligodendrocyte produces an estimated 5,000 mm2 membrane per day (Pfeiffer et al., 1993). These myelin sheaths are composed of membrane lipids and proteins all synthesized within oligodendrocytes (Fig. 1). Only cholesterol can be taken up from other cells if oligodendroglial biosynthesis is perturbed, which slows down myelination considerably (Saher et al., 2005). Where do metabolic precursors of myelin proteins and lipids come from? It is assumed that these molecules are largely derived from glucose transported to the brain by the blood and taken up from capillaries by endothelial cells. The import of glucose (but also essential fatty acids, vitamins, ions, etc.) and the subsequent transport steps of metabolites and their derivatives require the interaction of all cells involved, i.e. endothelial cells and astrocytes, astrocytes and oligodendrocytes, astrocytes and axons, oligodendrocytes and axons, and presumably by glial cells among themselves. One prerequisite for exchange of metabolites is the presence of appropriate transporters at the relevant cell surfaces, including glucose transporters (GLUT), monocarboxylate transporters (MCT), and connexins (Cx). While these transport processes are most likely relevant throughout life, gap junction mediated transport will be discussed here; exchange of glucose and lactate will be presented later in this review. Gap junctions, formed by connexins in plasma membranes of two adjacent cells, are cytoplasmic channels allowing exchange of small molecules and ions between these two cells (Goodenough and Paul, 2009). The main connexin isoforms of astrocytes are Cx30 and Cx43, while oligodendrocytes mainly express Cx32 and Cx47 (Cotrina and Nedergaard, 2012; Nualart-Marti et al., 2013; Theis and Giaume, 2012). In Cx47-deficient mice, myelin abnormalities include hypomyelination and vacuolation, indicating that generation and maintenance of myelin requires exchange of molecules via gap junctions. In human patients, developmental defects of CNS myelination (leukodystrophies) comprise rare genetic disorders, like Pelizaeus-Merzbacher disease (PMD, see below) and Pelizaeus-Merzbacher like disease (PMLD). These are defined by mutations of the PLP1 (PMD) and Gj12/Cx47 gene, respectively, leading to hypomyelination and secondary axonal loss, associated with retarded motor development, spasticity, and often premature death. Interestingly, a mouse line carrying the same Gj12/Cx47 point mutation reported in a PMLD patient exhibits reduced gap junction coupling between oligodendrocytes and myelin defects in juvenile but 1751

no longer in adult stages (Tress et al., 2011). This suggests that Cx47 contributes to the transport route of metabolites which oligodendrocytes require for efficient myelin synthesis. Vacuoles within myelin suggest that perturbed ionic and/or metabolic fluxes cause osmotic swellings that disrupt myelin membrane adhesion. Astrocytes are coupled to other astrocytes mainly by Cx43, while oligodendrocyte-oligodendrocyte gap junctions are composed of Cx32 (Cotrina and Nedergaard, 2012). In contrast, oligodendrocytes and astrocytes are coupled to each other by Cx47/Cx43 and Cx32/Cx30 heterotopic gap junctions (Cotrina and Nedergaard, 2012), implying that astrocyte-oligodendrocyte coupling will only be genetically impaired by loss of both Cx47 (or Cx43) and Cx30 (or Cx32) genes. Indeed, Cx32/Cx43 double mutant mice lack functional astrocyte-oligodendrocyte coupling and exhibit myelin vacuolation and early mortality (Magnotti et al., 2011). Similarly, oligodendrocyte to oligodendrocyte coupling was maintained in Cx47/Cx30 double mutant mice, but coupling between astrocytes and oligodendrocytes was completely disrupted resulting in dysmyelination and motor impairments (Tress et al., 2012). Assuming that this pathology reflects an impairment of astrocyte-oligodendrocyte coupling and reduced access to glucose (or glucose products), the significant amount of myelin still made suggests that oligodendrocytes import glucose also directly from the extracellular milieu. Gap junctions have been considered “unselective” channels allowing passage of molecules smaller than 1 to 2 kDa (Bruzzone et al., 1996). However, gap junctions show some selectivity (Harris, 2007), e.g. for metabolites as reported for cultured astrocytes. Glyceraldehyde-3-phosphate, NADH and NADPH readily traffic through gap junctions into neighboring cells, whereas glucose-6-phosphate does not (Gandhi et al., 2009), but the underlying mechanisms are not understood. Whether gap junctions between astrocytes and oligodendrocytes have similar selective properties, and whether this selectivity also occurs in vivo and is subject to regulation, is not known.

Synthesis of Myelin: Production of NADPH for Fatty Acid Synthesis During myelination oligodendrocytes synthesize huge amounts of lipids for integration into the growing myelin. Besides acetyl-CoA as carbon source, reduction equivalents (NADPH) are needed for fatty acid synthesis. NADPH is generated from NADP1 in the cytosol by four enzymatic reactions. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are part of the pentose phosphate pathway (PPP). Cytosolic NADP1dependent isocitrate dehydrogenase (ICDH) and the cytosolic malic enzyme (ME) reduce NADP1 to NADPH (Hirrlinger 1752

and Dringen, 2010) ICDH and ME also have mitochondrial isoforms not considered further here as fatty acid synthesis takes place in the cytosol. The PPP is the major metabolic pathway for production of cytosolic NAPDH (Dringen et al., 2007) and all components are present in oligodendrocytes. G6PDH is enriched in rat spinal cord oligodendrocytes (Kugler, 1994). Its activity was detected in cultured cells (Rust et al., 1991), in acutely isolated oligodendrocytes and myelin from rat brain (Cammer et al., 1982). Also 6PGDH has been found in cultured oligodendrocytes (Rust et al., 1991). Immunohistochemical analyses of human brain sections and murine cell cultures showed expression of transaldolase, an enzyme of the non-oxidative part of the PPP, in oligodendrocytes (Banki et al., 1994). Finally, transketolase is expressed in oligodendrocytes of rat and human brain and has been identified as a potential autoantigen in multiple sclerosis (Calingasan et al., 1995; Lovato et al., 2008). Collectively, these data suggest that oligodendrocytes are capable of generating NADPH via the PPP, consistent with the substantial flux of glucose through the PPP in cultured oligodendrocytes (Edmond et al., 1987). In cultured brain cells, activity of ICDH has been reported (Minich et al., 2003). While in astrocytes and microglia 50% each of ICDH activity was found in the cytosol and mitochondria, in oligodendrocytes (and neurons) 75% of its activity was localized to the cytosol (Minich et al., 2003), consistent with the concept that oligodendrocytes use ICDH intensively for generation of NADPH within the cytosol. Furthermore, cytosolic ME was detected by immunohistochemistry in cultured oligodendrocytes, but data on celltype specific expression in vivo is still lacking (Kurz et al., 1993). However, in chick spinal cord the activity of malic enzyme increased shortly preceeding onset of myelination, which was interpreted as a hint for an important role of this enzyme for lipid synthesis for myelination (Burt, 1971). Taken together, oligodendrocytes are equipped with a number of mechanisms to cope with the NADPH demand necessary for lipid synthesis during myelination. In addition, NADPH is needed in oligodendrocytes even after completion of myelination for other functions including glutathionebased defense against oxidative stress (Hirrlinger et al., 2002).

Synthesis of Myelin: The Limiting Role of Cholesterol A well-studied example how the rate of oligodendroglial metabolism determines development of myelin is cholesterol biosynthesis. Myelin is highly enriched in cholesterol and 80% of adult brain cholesterol resides in myelin (Dietschy, 2009; Muse et al., 2001; Saher et al., 2011). Although cholesterol is taken up from the diet and made de novo in liver, an intact blood–brain barrier prevents cholesterol import into Volume 62, No. 11

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the CNS (Bjorkhem and Meaney, 2004). Thus, cholesterol required for myelin biogenesis has to be synthesized in situ. Mice with an oligodendrocyte-specific deletion of the gene for squalene synthase (Fdft1), catalyzing the first committing step of cholesterol biosynthesis, show strongly impaired CNS myelination reflecting a severe delay of normal development (Saher et al., 2005). Horizontal transfer of cholesterol from genetically wild type cells allowed mutant oligodendrocytes to accumulate cholesterol over time. Importantly, the rate of myelination was dictated by cholesterol import and was thus very slow. In addition, mRNAs encoding major myelin proteins were down regulated, implying coordinated regulation of cholesterol levels and myelin gene expression. Surprisingly, myelin membranes purified from these mutant brains exhibited “normal” (i.e. high) levels of cholesterol. Thus, oligodendrocytes not only can take up and enrich cholesterol from outside sources, they also control the “cholesterol to other lipid” ratio in membranes destined to be incorporated into myelin (Saher et al., 2005). Where does cholesterol come from? Astrocytes are known to support synaptogenesis by releasing cholesterol (Mauch et al., 2001; Pfrieger, 2003) and are probably the source of cholesterol for squalene synthase deficient oligodendrocytes (Saher et al., 2005). Cholesterol can be rate-limiting for myelination also in other white matter diseases. Mice with transgenic overexpression of the proteolipid protein gene (Plp1) model human Pelizaeus-Merzbacher disease (PMD, see above), a leukodystrophy often caused by duplication of the PLP1 gene (Readhead et al., 1994). In oligodendrocytes of these mice, proteolipid protein and cholesterol lack their normal ratio and accumulate in the endo/lysosomal compartment (Simons et al., 2002) where they further block cholesterol biosynthesis (Saher et al., 2012). The perturbed blood-brain-barrier in this disease model allows cholesterol from the circulation to reach the brain. Indeed, feeding Plp1 transgenic mice a cholesterol-enriched diet ameliorates PMD-like pathology, increases myelin content, and even improves motor deficits (Saher et al., 2012). This highlights the crucial role of cholesterol metabolism for formation of myelin and suggests therapeutic options for patients suffering from PMD, provided that extrinsic cholesterol likewise reaches PLP1 overexpressing oligodendrocytes in vivo.

“Energy Metabolism” and Maintaining Axonal Functions After completion of myelination, a major task for glial cells in white matter tracts is the provision of energy-rich substrates to axons, required for fast axonal transport and propagation of action potentials (Fig. 2). Glucose (or glucose-6phosphate derived from glycogen) and the glycolysis products pyruvate and lactate are the major metabolites for this transfer. The axonal compartment in a myelinated nerve has only November 2014

limited access to glucose from the extracellular milieu at the nodes of Ranvier. The internodal space between axon and myelin is sealed by paranodal loops. While diffusion of metabolites underneath (and helically around) these septatelike junctions is in principle possible (Mierzwa et al., 2010), it is slow (taking many minutes) and likely insufficient to maintain rapid metabolic fluxes. In the nodal region many axons are in contact to astroglial processes and have additionally direct access to the extracellular milieu. However, it is unknown whether and to what extent neuronal glucose transporters are localized at the node of Ranvier. Transport of Energy Substrates Glucose is imported into cells by different passive glucose transporters (GLUT). GLUT1 is present on endothelial cells, astrocytes and oligodendrocytes, while neurons express mainly GLUT3 (Choeiri et al., 2002; Maher et al., 1994). However, glucose is unlikely to be released again from cells due to fast phosphorylation by hexokinase and lack of glucose-6phosphatase in brain. Instead, the glycolysis products pyruvate or lactate are exchanged between cells via monocarboxylate transporters (MCT; Fig. 2). Three isoforms (MCT1, 2, and 4) are expressed in brain and cotransport one proton and one monocarboxylate. They differ in cell type-specific expression and transport kinetics (Halestrap and Wilson, 2012). MCT2 is preferentially expressed in neurons, MCT4 is mainly astroglial, and MCT1 has been localized to astrocytes and oligodendrocytes (Lee et al., 2012; Pierre and Pellerin, 2005; Pierre et al., 2000; Rinholm et al., 2011). Contradicting the restricted localization of MCT1 to astrocytes based on immunohistochemistry (Pierre et al., 2000), recent in vivo observations in BAC transgenic mice suggest the transcription of MCT1 is specific to oligodendrocytes (Lee et al., 2012). While the localization of MCT1 is therefore still controversial, these MCT expression data indicate that pyruvate and lactate trafficking is a cell-type specific controlled process in white matter tracts, involving astrocytes, oligodendrocytes and axons. MCTs also transport ketone bodies and short fatty acids (Moschen et al., 2012; Pierre and Pellerin, 2005). Crucial determinants of metabolite exchange are the extra- and intracellular concentration and the pH gradient across the membrane. Extracellular glucose concentrations in grey matter were estimated by microdialysis between 0.5 mM and 2 mM (de Vries et al., 2003; McNay and Gold, 1999). However, due to the size of microdialysis capillaries these concentrations are spatially averaged. The real concentrations of metabolites in any microenvironment are unknown. This is especially relevant for the extracellular (periaxonal) 10 to 20 nm wide space between the axon and its surrounding myelin sheath. This volume is so small that release (or uptake) of only few molecules or protons can substantially 1753

change their local concentration. Also diffusion of metabolites within each cell can be a limiting factor for exchange. Again, this might be especially relevant for oligodendrocytes with their fine processes leading to the myelin sheath. Compact myelin imposes a diffusion barrier for intracellular ions and molecules between the adaxonal and abaxonal cytosolic spaces, leaving only non-compacted cytoplasmic (“myelinic”) channels, including the paranodal loops and Schmidt–Lanterman incisures, as potential routes for oligodendroglial cytosolic metabolites close to the axon (Nave, 2010b). Interestingly, adjacent layers of myelin are additionally interconnected at paranodal loops and Schmidt–Lanterman incisures by gap junctions consisting of Cx32 (Fig. 2). This direct coupling greatly reduces the distance and diffusion time between the oligodendrocyte soma and the adaxonal cytosolic space (Balice-Gordon et al., 1998; Kamasawa et al., 2005). The importance of these gap junctions for spatial buffering of K1 was suggested by genetic ablation of Cx32 and Cx47 (the oligodendroglial connexins), which caused osmotic problems and vacuolation of myelin upon higher neuronal activity (Menichella et al., 2006). It is likely but not proven that the same transport route is also accessible for the transfer of small metabolites. Glycogen Glycogen is the storage form of glucose and the highest amounts are found in liver and muscle. Albeit at much lower level, glycogen is present in the brain mainly in astrocytes both in grey and white matter (review: Brown and Ransom, 2007). However, recent evidence indicates that also neurons have an active glycogen metabolism (Saez et al., 2014). Synthesis of glycogen from glucose is energy consuming: (1) glucose is phosphorylated to glucose-6-phosphate by hexokinase using ATP as cosubstrate; (2) after conversion to glucose-1-phosphate by phosphoglucomutase, UDP-glucose pyrophosphorylase uses UTP to generate uridine diphosphate glucose (UDP-glucose), the final activated intermediate needed by glycogen synthase for chain elongation. Part of the energy invested in glycogen synthesis is retrieved during glycogen degradation. Glycogen is—with the exception of the branching points—degraded by phosphorolysis, leading via glucose-1-phosphate to glucose-6-phosphate, thereby bypassing the ATP-dependent reaction of hexokinase. For a long time it was assumed that glycogen in the brain is only an emergency fuel, e.g. during transient ischemia and other pathological conditions. However, there is compelling evidence that glycogen metabolism contributes actively to maintain energy balance also during physiological brain activity. For example, generalized sensory stimulation modulates the glycogen content of the brain (Dienel et al., 2002). Glycogenolysis is controlled by signaling molecules and ions like vasoactive 1754

intestinal polypeptide, noradrenaline, potassium and bicarbonate (Choi et al., 2012; Hof et al., 1988; Magistretti et al., 1981). Even, a “glycogen shunt” has been proposed suggesting that a fraction of glucose is first assembled into glycogen before being metabolized (Walls et al., 2009), consistent with a continuous turnover of glycogen (Dienel et al., 2007; Nelson et al., 1968; van Heeswijk et al., 2010; Watanabe and Passonneau, 1973). Indeed, astroglial glycogen metabolism is essential for normal higher brain functions, such as learning and memory formation (Gibbs et al., 2006; Newman et al., 2011; O’Dowd et al., 1994; Suzuki et al., 2011). The optic nerve contains ganglion cell axons, astrocytes and oligodendrocytes, but no neuronal somata. In a series of pioneering ex vivo experiments, it has been established that astroglial glycogen is essential for maintaining action potential firing rates in this white matter tract (for overview: Brown and Ransom, 2007; Ransom and Fern, 1997). It is thought that an “energy rich” metabolite derived from glycogen is transferred to the axon where ATP is generated. As glucose-6-phosphate (the degradation product of glycogen) cannot exit the cells and the enzyme glucose-6-phosphatase is not detectable in astrocytes, the critical metabolite is most likely not glucose. There is compelling evidence that white matter axons utilize either pyruvate or lactate. In ex vivo analyses of optic nerve function, blockade of monocarboxylate transporters inhibits the metabolic support of astroglial glycogen in maintaining axonal conductivity. Moreover, external lactate can substitute when the astroglial glycogen content is depleted (Brown et al., 2003; Tekkok et al., 2005). The exact route of lactate into the axonal compartment remains to be elucidated. Within white matter, astroglial processes (or free metabolites released from astrocytes into the extracellular space) have direct access to the axonal membrane only at the nodes of Ranvier, since the axonal internodal region is completely covered by myelin (Nave, 2010a, b). However, it is unknown whether the axonal membrane at the node of Ranvier, densely packed with ion channels and scaffolding proteins, also harbors MCT2 (or other transporters for metabolites). Theoretically, astroglial glycogen break-down products could be transferred to glycolytic oligodendrocytes and monocarboxylates (pyruvate or lactate) could be released along the periaxonal space; consistent with the finding that the internodal axonal membranes harbor MCT2 (Rinholm et al., 2011) and fully glycolytic oligodendrocytes can support axons (Funfschilling et al., 2012). Indeed, interfering with oligodendrocyte specific MCT1 gene expression results in axon damage and neuronal loss (Lee et al., 2012). These observations strongly suggest that oligodendrocytes contribute to the transfer of monocarboxylates towards the axon. Lactate Lactate has a long history in research on brain energy metabolism. In 1994, the astrocyte to neuron “lactate-shuttle” Volume 62, No. 11

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hypothesis was proposed for cortical grey matter. It suggested that glutamate uptake into perisynaptic astrocytes triggers glucose uptake, glycolysis, and the release of pyruvate and/or lactate, which are taken up by neurons to generate ATP by oxidative phosphorylation (Pellerin and Magistretti, 1994; Pellerin et al., 2007), establishing a metabolic “feedback” link of astrocytes to neuronal activity. Conceptually the “lactate shuttle” is firmly linked to glutamate reuptake at synapses in the cortical gray matter and has been extended to include glycogen as astroglial glucose reservoir that can be degraded and converted to lactate on demand (Dringen et al., 1993). Is there a similar “lactate shuttle” in white matter tracts and in the absence of synapses? Are oligodendrocytes involved in activity-dependent glucose utilization and lactate metabolism? Cultured oligodendrocytes can take up lactate from the medium for mitochondrial respiration and lipid synthesis (Sanchez-Abarca et al., 2001; Fig. 1). Also differentiating oligodendrocytes in slice cultures take up lactate and it has been suggested that imported lactate protects oligodendrocytes and their precursors during low glucose availability (Rinholm et al., 2011). However, the analysis of mutant mice suggests that oligodendrocytes in vivo are not consuming but are rather a source of lactate (Fig. 2). Mature oligodendrocytes in the CNS lacking mitochondrial respiration due to cell-type specific disruption of the Cox10 gene (encoding a farnesyl transferase required to assemble complex IV) are fully viable and support myelin and axonal integrity, most likely by releasing lactate generated by aerobic glycolysis (Funfschilling et al., 2012). Furthermore, an increase in brain lactate was observed in these mice in grey and white matter. Importantly, this lactate signal was only detectable under isoflurane anesthesia, an unspecific brake on mitochondrial respiration (Boretius et al., 2013), and dropped thereafter within minutes to undetectable levels in both mutants and controls (Funfschilling et al., 2012). Thus, under these experimental conditions mature oligodendrocytes survive by only glycolysis and are a source of lactate rapidly metabolized by other cells in white matter, including the axonal compartment (Funfschilling et al., 2012). This transfer of lactate from oligodendrocytes to axons is functionally important as evidenced by mice heterozygous for a null mutation of the Mct1 gene (or an oligodendrocyte-specific knockdown of MCT1). These show axonal swellings, Wallerian degeneration and neuronal loss (Lee et al., 2012). Interestingly, MCT1 is downregulated in patients with ALS, suggesting a role for diminishing supply of lactate from oligodendrocytes to axons in the pathology of this disease (Lee et al., 2012). We note that the published in vitro and in vivo data of oligodendroglial lactate metabolism refer to different stages of white matter development and may not be contradictory. In postnatal development myelinating oligodendrocytes perform November 2014

oxidative phosphorylation and might profit from circulating lactate as a rate-limiting resource and metabolite for lipid synthesis and energy production. At a later stage, oligodendrocytes appear to undergo a metabolic (“glycolytic”) switch and begin to release pyruvate and/or lactate themselves to support axonal functions (Funfschilling et al., 2012). This is reflected by the vulnerability of immature oligodendrocytes and their precursors to mitochondrial toxins prior to myelination (Ziabreva et al., 2010; unpublished data). Also germ line mutations of the human COX10 gene affecting OPC lineage cells prior to myelination cause a syndromic mitochondrial disease including a leukodystrophy (Antonicka et al., 2003). In contrast lack of functional mitochondria after completion of CNS myelination is well tolerated, presumably because switching to aerobic glycolysis is one of the last steps in oligodendrocyte differentiation. The mechanisms underlying such a developmental switch of oligodendroglial metabolism are not understood but they are reminiscent of the glycolytic switch observed in many tumors including gliomas (“Warburg effect”). It is tempting to speculate that the latter is the recruitment of a normal developmental program by tumor cells. Its elucidation will allow a deeper understanding of general principles how cells adapt their metabolism to changing requirements.

N-Acetyl-Aspartate: A Metabolite in Search of Functions Neuronal N-acetyl-aspartate (NAA) is with 10 mM (Henriksen, 1995) one of the most abundant metabolites in adult brain, but its physiological function has remained enigmatic. NAA can be quantified by 3H-magnetic resonance spectroscopy and is clinically often used as a marker for neuronal integrity. In white matter tracts, NAA is an axonal marker (Bjartmar et al., 2002). However, using immunelectron microscopy, NAA was localized to oligodendrocytes as well as axons showing enrichment within myelin (Nordengen et al., 2013). In neurodegenerative diseases, such as multiple sclerosis (Bjartmar et al., 2000; Cader et al., 2007), or following brain hypoperfusion (Nitkunan et al., 2008), NAA levels are significantly reduced. Interestingly, reduced NAA can be reversible (De Stefano et al., 1995), suggesting that diseaseassociated reductions of NAA are not necessarily caused by the physical loss of neurons or axons, but are more likely a biochemical marker of imminent neurodegeneration. Several potential functions of NAA have been proposed (for review: Moffett et al., 2007), but except for a supportive role in myelination none has been proven. NAA is synthesized in neurons by acetyl-CoA dependent acetylation of aspartate. While the NAA synthesizing enzymatic activity was localized to mitochondria (Ariyannur et al., 2008; Madhavarao et al., 2003; Patel and Clark, 1979), molecular cloning 1755

of this enzyme (NAT8L) and its subcellular localization could not yet confirm this (Ariyannur et al., 2010; Wiame et al., 2010). By immunostaining NAT8L resides in the endoplasmatic reticulum of cultured CHO cells and neurons (Wiame et al., 2010), consistent with one report detecting NAAsynthesizing activity mainly in the microsomal fraction (Lu et al., 2004). The localization of NAT8L has important consequences: its substrates aspartate and acetyl-CoA are readily available in mitochondria, whereas transport of acetyl-CoA into the ER requires additional enzymatic steps (“citrate shuttle”). NAA is degraded by the enzyme aspartoacylase (ASPA) mainly localized in oligodendrocytes (Klugmann et al., 2003), implying that NAA is preferentially shuttled from neurons to oligodendrocytes. ASPA may not be exclusive to oligodendrocytes: a subset of microglia and neurons could be immunostained for ASPA, while no expression was found in astrocytes (Moffett et al., 2011). Patients with Canavans disease, a severe leukodystrophy caused by loss-of-function mutations in the human ASPA gene, exhibit severe dysmyelination and vacuolar splitting of myelin membranes, the latter due to exceedingly high NAA levels and osmolarity problems. This observation was confirmed in corresponding Aspa mutant mice, where a similar neuropathology includes white matter vacuolation (Matalon et al., 2000), associated with lowered acetate levels and defective lipid synthesis in the brain (Madhavarao et al., 2005). The physiological function of NAA has remained an open question. Based on its abundance in the millimolar range, NAA was suggested to serve as an “osmoregulator” (Moffett et al., 2007; Taylor et al., 1995). Experimental evidence, including the phenotype of Aspa and Aralar mutant mice (Matalon et al., 2000; Ramos et al., 2011), supports the idea that neuronal NAA provides acetate residues to oligodendrocytes for synthesis of myelin lipids (Hagenfeldt et al., 1987). Indeed, acetate from NAA is preferentially integrated into myelin (Burri et al., 1991; Fig. 1). However, a NAT8Ldeficient mouse (lacking the NAA synthesizing enzyme) has been generated in several laboratories and is viable without obvious signs of dysmyelination (Tzvetanova et al., unpublished data). This mutant shows deficits in social interaction and altered expression of several neurotrophic factors and cytokines (Furukawa-Hibi et al., 2012), indicating that the function of NAA might be more complex and might change between developing to adult organisms. The origin and fate of the aspartate moiety of NAA is another unresolved aspect of NAA metabolism (Fig. 2). Neurons synthesize NAA from aspartate, potentially depleting the pool of aspartate and/or oxaloacetate. On the other hand, it has been proposed that neurons can operate a “truncated” TCA cycle, using glutamine or glutamate as substrate (Cam1756

bron et al., 2012). In this scenario, formation of NAA from aspartate favors conversion of glutamate to alphaketoglutarate to fuel the cycle and to support neuronal/axonal energy production (Cambron et al., 2012). Furthermore, the fate of aspartate in oligodendrocytes, once released from NAA, is also unclear. Aspartate could be converted to acetylCoA (via oxaloacate, malate and pyruvate). Alternatively, aspartate is metabolized in mitochondria, used for protein synthesis or is shuttled back into the axonal compartment. The latter is less likely based on an earlier N-([2H3]acetyl)-L[15N]aspartate in vivo labeling study (Miller et al., 1996). Several lines of evidence indicate an important interaction of neurons and oligodendrocytes in NAA metabolism. However, also astrocytes might be involved. While neither NAT8L, ASPA, nor NAA has been localized to astrocytes, they metabolize N-acetyl-aspartyl-glutamate (NAAG) synthesized by neurons from NAA. NAAG acts as a ligand on NMDA receptors on neurons and oligodendrocytes (Kolodziejczyk et al., 2009; but see Losi et al., 2004) and also activates the metabotropic glutamate receptor mGluR3 (Wroblewska et al., 1997). NAAG is hydrolyzed to NAA and glutamate (which will itself activate glutamate receptors) by glutamate-carboxy-peptidase-II (GCPII) located on the surface of astrocytes (Berger et al., 1999; Neale et al., 2011). GCPII immunoreactivity has also been described in white matter (Sacha et al., 2007), and its mRNA is more abundant in myelinating oligodendrocytes compared to oligodendrocyte precursors (Cahoy et al., 2008; de Monasterio-Schrader et al., 2012), suggesting that also oligodendrocytes are able to degrade NAAG. NAA produced in this way is likely taken up by oligodendrocytes and degraded. In a patient with Pelizaeus-Merzbacher-like-Disease (PMLD), a leukodystrophy caused by the mutation of the gap junction gene Cx47, an elevated level of NAAG has been reported (Mochel et al., 2010; Sartori et al., 2008). How mutations in this gene affect NAAG steady state levels remains to be established.

Regulation of Metabolism and Metabolic Interaction in CNS White Matter Compelling evidence indicates that metabolites are exchanged between different types of cells and their processes within white matter tracts, i.e. the axonal compartments, the myelin sheath and its cytosolic channel system, oligodendrocytes and astrocytes, the latter contacting both nodes of Ranvier and blood vessels (Saab et al., 2013). If this model is true, it is reasonable to assume that metabolic interactions are also tightly regulated and adjusted to different physiological states. For example, during maturation of white matter tracts the coordination in the supply of lipids and lipid precursors with the formation of myelin is essential. Later, axonal tracts firing action potentials at high frequencies most likely request a Volume 62, No. 11

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higher degree of metabolic support from neighboring glial cells. On the other hand exceeding the release of lactate beyond current needs might quickly cause pH changes and be detrimental to axon function. However, the regulatory signals exchanged between the different cellular compartments remain to be elucidated. Which molecules are candidates for metabolic signaling? Besides being a substrate in energy metabolism, lactate can act as a signaling molecule (Barros, 2013; Bergersen and Gjedde, 2012). This idea was strengthened by the discovery that Gprotein coupled receptor 81 (GPR81; also known as HCAR1) is a lactate receptor (Cai et al., 2008; Liu et al., 2009). In grey matter, GPR81 has been localized to neurons and astrocytes with enrichment at excitatory synapses (Lauritzen et al., 2013). However, GPR81 expression in white matter has not been clarified. Furthermore, lactate is involved in blood flow regulation (Gordon et al., 2008), the modulation of Ca21-signals and of intracellular free glucose in astrocytes (Requardt et al., 2012; Sotelo-Hitschfeld et al., 2012). A number of channels and transporters are modulated by lactate or by lactate induced changes in the NAD1/NADH-ratio (Barros, 2013), a central redox pair in the regulation of glial metabolism (Hirrlinger and Dringen, 2010; Wilhelm and Hirrlinger, 2012). In oligodendrocytes, signaling via the NAD1-dependent deacetylase sirtuin 2 (SIRT2) is a potential mediator of metabolic regulation. While SIRT2 is present in oligodendrocytes and CNS myelin (Werner et al., 2007), its downstream targets are not yet elucidated. Another potential axon-to-oligodendrocyte signaling molecule is NAAG. In invertebrates (crayfish) NAAG is released from axons during electrical stimulation (Urazaev et al., 2001). As mentioned, NAAG can activate glutamate receptors either directly or after releasing glutamate by cleavage with GCPII. The expression of different types of glutamate receptors on oligodendrocytes is well documented, although most studies have concentrated on pathological situations rather than physiological functions (Berger et al., 1992; Karadottir et al., 2005; Kolodziejczyk et al., 2010; Micu et al., 2006; Patneau et al., 1994). These receptors might also be directly activated by glutamate released from axons (Kukley et al., 2007; Ziskin et al., 2007). Whether oligodendroglial glutamate receptors participate in the coordination of energy metabolism in white matter is currently under investigation. The beauty of NAAG as a signaling molecule is that (besides activating receptors) it comes with potential substrates for energy production, i.e. the acetate and aspartate residues in the NAA moiety of NAAG. During action potential propagation, Na1 enters axons through voltage-gated sodium channels at the node of Ranvier. For repolarization, K1 is released through potassium channels localized at the juxtaparanodal and internodal regions, i.e. under the myelin sheath (Rash, 2010). Given the small periaxonal space underneath myelin, already the release November 2014

of small amounts of potassium will substantially increase K1concentration in this compartment and depolarize the membrane of the adjacent oligodendrocytes to up to 175 mV (David et al., 1992, 1993). This allows an effective K1siphoning away from the axon and prevents depolarization and uncontrolled action potential firing (Kofuji and Newman, 2004; Wallraff et al., 2006), but could also serve as a signal for oligodendrocytes that the axon propagates action potentials and requires an increased supply of energy-rich metabolites. In astrocytes, glycolysis is stimulated by increased extracellular K1 (Bittner et al., 2011). As the concentration of K1 in the extracellular space increases during action potential propagation in the axon, a similar mechanism in oligodendrocytes would lead to increased glycolysis rates and probably increased lactate release towards the axon.

Conclusions CNS white matter serves specialized functions, like reliable conduction of axonal action potentials and ATP-driven axonal transport, which imposes particular requirements on its metabolism. So far, most findings regarding regulation of brain metabolism were derived from analyzing grey matter regions, and little is known whether these mechanisms apply likewise to metabolic control in white matter tracts. Moreover, the metabolic demands change during development, from the synthesis of myelin to adult stages, when electrically and metabolically isolated axons require support. Recent evidence strongly suggests that both oligodendrocytes and astrocytes contribute to these tasks. Metabolites and signals have to be exchanged between axons, oligodendrocytes, the myelin compartment, and astrocytes, involving numerous potential transport routes for metabolites, both between and within cells. Cholesterol synthesized within oligodendrocytes is rate-limiting for myelin formation, while lactate metabolism probably changes during development. Several molecules, including lactate and NAA/NAAG, are potential signaling molecules involved in coordinating metabolism between different cell types within white matter. While an integrated view on CNS white matter metabolism has slowly emerged, many crucial questions are still unresolved: Are other energy metabolites than lactate exchanged between axons and glial cells and which route do they take? Is the metabolic cooperation of axons and oligodendrocytes regulated in an activity dependent manner? If so, which are the signaling molecules and mechanisms involved? What are the physiological functions of NAA? Which mechanisms regulate the metabolic switch during development? Answering these questions will lead to a better understanding of white matter metabolism and physiology and hopefully contribute to the development of therapeutic strategies aimed at myelin diseases and related neurodegenerative disorders.

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Black JA, Waxman SG. 1998. The perinodal astrocyte. Glia 1:169–183.

Acknowledgment Grant sponsor: DFG; Grant number: Hi1414/1-1 and Hi1414/2-1; Grant sponsor: German Diabetes Society (to J.H.), DFG Research Center “Nanoscale Microscopy and Molecular Physiology of the Brain”; Grant number: SFBTR43 and EU-FP7 (Leukotreat, Ngidd); Grant sponsor: BMBF (Leukonet), Oliver’s Army, and an ERC Advanced Grant (AxoGlia) (to K.A.N.). The authors apologize to many colleagues whose work could not be cited owing to space restrictions. The authors thank D. Attwell, S. Boretius, J. Edgar, H. Ehrenreich, S. Goebbels, B. Hamprecht, C. Kassmann, K. Kusch, A. Saab, G. Saher, M. Simons, I. Tzvetanova, and H. Werner for frequent discussions.

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