Oligodendroglial NMDA receptors regulate glucose import and axonal energy metabolism

“GLUT1 mRNA is more abundant in mature oligodendrocytes than in precursor cells (Zhang et al., 2014).”

“Some of the mice also carried the R26-stop-EYFP reporter gene for Cre activity (RRID: IMSR_ JAX:006148) (Srinivas et al., 2001).”

“, 2003) were crossbred to floxed NR1 (gene symbol Grin1) mice (RRID: MGI_2175051) (Tsien et al., 1996).”

“This confirms that axons readily use lactate for ATP generation (Fünfschilling et al., 2012; Lee et al., 2012; Tekkök et al., 2005; Wyss et al., 2011).”

“This confirms that axons readily use lactate for ATP generation (Fünfschilling et al., 2012; Lee et al., 2012; Tekkök et al., 2005; Wyss et al., 2011).”

“…signaling in oligodendrocyte differentiation and promoting myelinationhasbeensuggested, basedon in vitroobservations (Li et al., 2013; Lundgaard et al., 2013), which is compatible with neuronal activity stimulating OPC proliferation and myelination in vivo (Gibson et al., 2014; Liu et al., 2012).”

“Myelin was purified as described before (Norton and Poduslo, 1973) from mouse brain homogenate in 0.32 M sucrose.”

“3% by negative selection immunopanning (Barres et al., 1992).”

“Oligodendroglial metabolic support can explain why myelinated axons are vitally dependent on myelin integrity, independent of the myelin function in saltatory conduction (Griffiths et al., 1998; Kassmann et al., 2007; Lappe-Siefke et al., 2003).”

“Oligodendroglial metabolic support can explain why myelinated axons are vitally dependent on myelin integrity, independent of the myelin function in saltatory conduction (Griffiths et al., 1998; Kassmann et al., 2007; Lappe-Siefke et al., 2003).”

“While NMDA receptors are widely studied in glutamatergic neurotransmission, the role of NMDA receptors in oligodendrocytes has remained enigmatic (De Biase et al., 2011; Guo et al., 2012; Káradóttir et al., 2005; Li et al., 2013; Lipton, 2006; Lundgaard et al., 2013; Micu et al., 2006; Piña-Crespo et al., 2010; Salter…”

“…signaling in oligodendrocyte differentiation and promoting myelinationhasbeensuggested, basedon in vitroobservations (Li et al., 2013; Lundgaard et al., 2013), which is compatible with neuronal activity stimulating OPC proliferation and myelination in vivo (Gibson et al., 2014; Liu et al., 2012).”

“Oligodendrocytes, best known for making myelin (Emery, 2010; Nave and Werner, 2014), also support the survival of myelinated axons (Griffiths et al.”

“Importantly, axonal recovery after OGD was normal prior to age P10 (Figure S6A), suggesting that axons require oligodendroglial support mostly after myelination, which limits rapid axonal access to extracellular metabolites (Nave, 2010).”

“They have direct access to the vasculature, store glycogen, and rapidly respond to en- ergy deprivation with glycogen breakdown (Brown and Ransom, 2007).”

“is associated with NR2C and NR3A subunits that provide a weak Mg block (Burzomato et al., 2010; Das et al., 1998), suggesting that calcium entry does not require prior membrane depolarization.”

“While NMDA receptors are widely studied in glutamatergic neurotransmission, the role of NMDA receptors in oligodendrocytes has remained enigmatic (De Biase et al., 2011; Guo et al., 2012; Káradóttir et al., 2005; Li et al., 2013; Lipton, 2006; Lundgaard et al., 2013; Micu et al., 2006; Piña-Crespo et al., 2010; Salter…”

“Oligodendroglial NMDA receptors are localized in special- ized microdomains of myelinating processes (Stirling and Stys, 2010; Stys, 2011) and also face the periaxonal space (Micu et al., 2006).”

“…were separated on 10% SDS-polyacrylamide gels, blotted onto polyvinylidene difluoride membranes (Roche), and incubated with primary antibodies for GLUT1 (1:500, Abcam Cat# ab32551 RRID: AB_732605), MCT1 (1:1,000, Millipore Cat# AB1286 RRID: AB_90565), and SIRT2 (1:2,000) (Dryden et al., 2003).”

“Like unmyelinated axons (Kukley et al., 2007;Wakeet al., 2011; Ziskin et al., 2007) alsomyelinated axons release glutamate upon spiking, which induces calcium elevations in myelin (Micu et al.”

“Like unmyelinated axons (Kukley et al., 2007;Wakeet al., 2011; Ziskin et al., 2007) alsomyelinated axons release glutamate upon spiking, which induces calcium elevations in myelin (Micu et al.”

“Myelin is assembled as a spiral extension of oligo- dendroglial cell membrane, which requires vesicular transport of membrane material through non-compacted cytosolic chan- nels to the growing tip (Snaidero et al., 2014).”

“This confirms that axons readily use lactate for ATP generation (Fünfschilling et al., 2012; Lee et al., 2012; Tekkök et al., 2005; Wyss et al., 2011).”

“Glucose is amajor carbon source for lipid precursor metabolites, which are rate-limiting for myelination (Rinholm et al., 2011). ptake by Cultured Oligodendrocytes ulation mobilizes GLUT1 and increases its cell surface expression, which is panned NMDA-treated oligodendrocytes and controls. ea of n…”

“Oligodendroglial metabolic support can explain why myelinated axons are vitally dependent on myelin integrity, independent of the myelin function in saltatory conduction (Griffiths et al., 1998; Kassmann et al., 2007; Lappe-Siefke et al., 2003).”

“The developmental delay of myelination in NR1 mutant optic nerves fits well the unex- plained observation that demyelinating lesions repair less well when NMDA receptors are blocked (Li et al., 2013; Lundgaard et al., 2013).”

“However, the concept of glycolytic support by oligodendrocytes (Morrison et al., 2013; Saab et al., 2013) rai- ses the question of how glucose utilization is quantitatively regu- lated.”

“To determine functional GLUT1 insertion, we expressed a glucose sensor (Ta- kanaga et al., 2008) in transfected oligodendrocytes (Figure 1E).”

“126 Neuron 91, 119–132, July 6, 2016 The majority of myelinated axons in cortico-spinal tracts fire at a high frequency and comprise a high energy demanding white matter tract (Perge et al., 2012).”

“However, at P10, when many oligodendrocytes had matured and express Cnp1, about one-third of the floxed alleles were excised, and recombination reached 62% ± 8% at P96, when myelination is completed, matching the expected proportion of oligodendrocyte in adult optic nerves (Miller et al., 1985).”

“In development, NMDA receptors are most abundant in oligodendrocyte precursors (De Biase et al., 2010; Zhang et al., 2014).”

“Immunoblotting was performed as described before (Werner et al., 2007).”

“However, the concept of glycolytic support by oligodendrocytes (Morrison et al., 2013; Saab et al., 2013) rai- ses the question of how glucose utilization is quantitatively regu- lated.”

“This confirms that axons readily use lactate for ATP generation (Fünfschilling et al., 2012; Lee et al., 2012; Tekkök et al., 2005; Wyss et al., 2011).”

“130 Neuron 91, 119–132, July 6, 2016 CAP area is proportional to the total number of excited axons (Stys et al., 1991).”

“In the rodent optic nerve, the highest myelination rate occurs during the third postnatal week (Hildebrand and Waxman, 1984) and predicts the highest demands for glucose as a carbon source for lipid synthesis at this time.”

“During OGD, abnormal Ca entry into the axonal compartment triggers axon loss probably resulting from excessive glutamate signaling caused by reversal of glutamate transporters (Stirling and Stys, 2010; Tekkök et al., 2007).”

“Unfortunately, the early functions of glutamate signaling were not revealed by the deletion of NR1 in OPC (De Biase et al., 2011; Guo et al., 2012).”

“, 2006; Salter and Fern, 2005) may not be the main mechanism causing myelin damage (Hamilton et al., 2016).”

“For control experiments and sensor calibration (Bittner et al., 2010), cells were incubated without glucose or adding either iodoacetate (glycolysis inhibitor, 1 mM) or cytochalasin B (glucose transporter blocker, 40 mM).”

“Recently, axonal activity was shown to induce NMDA receptor-mediated calcium increases in myelin, indicating that adult oligodendrocytes have functional NMDA receptors (Micu et al., 2016).”

“A role for NMDA re- ceptor signaling in oligodendrocyte differentiation and promoting myelinationhasbeensuggested, basedon in vitroobservations (Li et al., 2013; Lundgaard et al., 2013), which is compatible with neuronal activity stimulating OPC proliferation and myelination in vivo (Gibson et al.,…”

“For oxygen-glucose deprivation (OGD), the protocol was performed as described previously (Baltan et al., 2008).”

“Next, we examined myelinated optic nerves under metabolic stress for their ability to recover from transient oxygen-glucose deprivation (OGD), a well-established model of acute ischemia (Tekkök et al., 2007).”

“In adipocytes, insu- lin-dependent GLUT4 trafficking requires calcium-depen- dent myosin motors (Yip et al., 2008).”

“Is NMDA receptor-dependent regulation of GLUT1 in oligodendrocytes and the non-compacted myelin compartment the same as GLUT4 regulation in other cell types (Bogan and Kandror, 2010)? In adipocytes, insulin-dependent GLUT4 trafficking requires calcium-dependent myosin motors (Yip et al.”

“Optic nerves were acutely removed and either immersion-fixed for conventional sample preparation or cryofixed by HPF and freeze substituted as described in detail (Möbius et al., 2010).”

“Cultured neurons mobilize glucose transporter GLUT3 in response to glutamate (Ferreira et al., 2011).”

“While NMDA receptors are widely studied in glutamatergic neurotransmission, the role of NMDA receptors in oligodendrocytes has remained enigmatic (De Biase et al., 2011; Guo et al., 2012; Káradóttir et al., 2005; Li et al., 2013; Lipton, 2006; Lundgaard et al., 2013; Micu et al., 2006; Piña-Crespo et al., 2010; Salter…”

“While NMDA receptors are widely studied in glutamatergic neurotransmission, the role of NMDA receptors in oligodendrocytes has remained enigmatic (De Biase et al., 2011; Guo et al., 2012; Káradóttir et al., 2005; Li et al., 2013; Lipton, 2006; Lundgaard et al., 2013; Micu et al., 2006; Piña-Crespo et al., 2010; Salter…”

“Cell Culture Preparation Primary mouse or rat oligodendrocyte progenitor cells (OPCs) were isolated from cerebral cortices at P0–P2 as described previously (Barros et al., 2009; McCarthy and de Vellis, 1980).”

“Here, we found no difference in the onset of EAE symptoms or in clinical severity (Figures S5B–S5D), as indicated earlier (Guo et al., 2012; Matute, 2010).”

“is associated with NR2C and NR3A subunits that provide a weak Mg block (Burzomato et al., 2010; Das et al., 1998), suggesting that calcium entry does not require prior membrane depolarization.”

“This suggests that NMDA receptors are not essential for myelination per se, in agreement with earlier reports (De Biase et al., 2011; Guo et al., 2012).”

“Oligodendroglial NMDA receptors are localized in specialized microdomains of myelinating processes (Stirling and Stys, 2010; Stys, 2011) and also face the periaxonal space (Micu et al.”

“Since the average spike count rate is the key determinant of axonal ATP consumption, oligodendrocytes need to ‘‘know’’ the spiking activity of myelinated axons, which can differ greatly in vivo (Barry, 2015; Perge et al., 2012).”

“While NMDA receptors are widely studied in glutamatergic neurotransmission, the role of NMDA receptors in oligodendrocytes has remained enigmatic (De Biase et al., 2011; Guo et al., 2012; Káradóttir et al., 2005; Li et al., 2013; Lipton, 2006; Lundgaard et al., 2013; Micu et al., 2006; Piña-Crespo et al., 2010; Salter…”

“Oligodendrocytes appear to sense the metabolic demands of axons by N-methyl-D-aspartate (NMDA) receptors that are associated with the internodal/paranodal membrane (538).”

“Either glucose or lactate can be used as an energy source for white matter (Brown et al., 2001; Saab et al., 2016), and glial glycogen can supply energy to axons during aglycemia (Brown et al.”

“During development, OPCs give rise to oligodendrocytes that generate myelin, which ensures fast signal transmission and provides metabolic support to axons (Nave, 2010; Saab et al., 2016).”

“Alternatively, one could try to enhance the shuttling of metabolites through connexin gap junctions, or enhance NMDA signaling in oligodendrocytes to increase glucose uptake through oligodendrocyte GLUT1.”

“…WMI are mainly associated with impaired cognitive functioning later in life are essential for proper brain connectivity, as myelination enables rapid and efficient propagation of action potentials, and provides protection and trophic support to axons (Funfschilling et al., 2012; Saab et al., 2016).”

“, monocarboxylate transporter 1) are located on its adaxonal surface (Rinholm et al., 2011; Lee et al., 2012b; Nijland et al., 2014; Domenech-Estevez et al., 2015; Saab et al., 2016).”

“Previously, myelin has been regarded as an inert and purely insulating membrane, but it is now clear that myelin is metabolically active, providing support to the underlying axon (F€unfschilling et al., 2012; Lee et al., 2012; Saab et al., 2016).”

“The facilitated glucose transporter 1 (GLUT1) mediates glucose diffusion from endothelial cells and into astrocytes and oligodendrocytes (300, 379).”

“Myelinating oligodendrocytes can alter action potential propagation speed by regulating potassium levels in the periaxonal space (48) and changing the expression and distribution of molecules involved inmetabolic support of the axon, potentially relevant in setting its maximum firing rate (2, 24).”

“Over the past 20 years, microglia have been shown to have important functions in regulating the health of myelin, the lipid-rich membrane that surrounds neuronal axons that ensures the rapid propagation of electrical impulses and supports axonal metabolic demands 11,12 .”

“Moreover, PV+ interneurons are highly energy demanding (Kann et al. 2014), for which myelin provides axonal metabolic support (Fünfschilling et al. 2012; Lee et al. 2012b; Saab et al. 2016).”

“Myelinating oligodendrocytes that are existent behind the optic nerve head also provide metabolic support to axons, besides maintaining the axonal integrity (Saab et al., 2016).”

“Finally, NMDA glutamate receptors localized in the myelin sheath (Micu et al., 2006, 2016; Saab et al., 2016) can enhance myelination in cell culture (Wake et al.”

“Moreover, the ensheathing OLs can transfer energy substrates into axons (Fünfschilling et al., 2012; Lee et al., 2012b; Saab et al., 2016) possibly supporting them during periods of high metabolic demand.”

“The CAP area is proportional to the total number of excited axons and represents a convenient and reliable means of monitoring optic nerve axon function (Stys et al., 1991; Saab et al., 2016).”

“OLs are myelin-forming cells in the CNS and provide metabolic support to neurons by forming myelin sheaths around axons (Saab et al., 2016).”

“Recent work from Nave and colleagues further suggests that the mechanism by which glycolysis in oligodendrocytes is matched to different axonal energy needs involves the release of glutamate through axonal activity [46].”

“Indeed, stimulation of NMDARs, expressed in immature and mature oligodendrocytes, promote the maturation of these cells and myelination around axons [181–183], upregulates their energy metabolism, increases mitochondria motility within myelin sheath, and glycolytic support to the axons [184, 185].”

“Myelinating oligodendrocytes also provide fast spiking axons with lactate or pyruvate 2–4 for the generation of ATP 5 .”

“OLs appear to generate lactate and pyruvate in an activity-dependent manner: glutamate release from neurons activates NMDA receptors on OLs, increasing glucose uptake and lactate production through glycolysis (Saab et al., 2016).”

“…through oligodendrogenesis and remyelination with the bulk of remyelination occurring within two weeks in focal chemical models of demyelination (Duncan G. J. et al., 2017) and within two months in cuprizone demyelination (Sachs et al., 2014) and oligodendrocyte depletion (Traka et al., 2010).”

“Indeed, disruption of the axon–myelin unit can occur acutely, such as during transient ischaemia, or chronically in MS and other neurodegenerative diseases, and involve myelinic NMDA receptors, a key component regulating the supply of energy substrates between oligodendrocyte/myelin and axons [134].”

“Glutamate receptor activation may increase OPC energy supply, promoting their survival, proliferation or differentiation [75,76].”

“, 2016), whereby NMDA receptors, known to be localized to the myelinating processes of oligodendrocytes (Káradóttir & Attwell, 2007), can respond to axonal glutamatergic release and trigger the redistribution of glucose transporters within the myelinating cell to regulate the metabolic support pathways mediated by the…”

“…in dendritic or presynaptic compartments of neurons, or in axons, particularly those in white matter that are surrounded by myelin and seem quite likely to rely on surrounding glial cells (oligodendrocytes) for metabolic support (Meyer et al., 2018; Saab et al., 2016; Trevisiol et al., 2017).”

“Glutamatergic signaling has been shown to stimulate the surface expression of glucose transporter 1 (GLUT1) in OLs, suggesting that axonal activity might regulate the metabolic support provided by OLs to axons 10 .”

“Glucose or lactate is provided to axons through astrocytes or oligodendrocytes (Saab et al., 2016) via glucose and monocarboxylate transporters (MCTs), respectively (Pierre et al., 2002; Simpson et al., 2007).”

“The extent to which intercellular coupling is reduced inwhitematter tractsof cKOanimals remains tobedetermined, and follow-up studies could further resolve whether alterations in panglial coupling impact axonal physiology and energy homeostasis (Looser et al., 2018; Saab et al., 2016).”

“…from defective axon-glia interactions is in agreement with findings in oligodendroglial mutants 10–13 , and transplantation experiments 14,21,24,25 and is consistent with our understanding of oligodendroglial-mediated mechanisms, such as axonal metabolic support 7–9,26,27 and detoxification 28 .”

“The crucial role of MCT1, a mover of energy substrate from the oligodendroglial compartment to axons, in axon function and survival spurred investigation into the regulation of energy exchange between oligodendrocyte and neuron (Saab et al., 2016).”

“They use lactate for their own energy needs and also supply neighbouring axons with lactate [42].”

“Then, glucose is transported into neurons through GLUT3 and GLUT4 [11–14], or imported into oligodendrocytes and microglia through GLUT1 [15, 16]”

“Notably, RGC axons can also receive energy metabolites, particularly lactate, from neighboring glial cells like astrocytes and oligodendrocytes (Brown et al., 2003; Saab et al., 2016; Wender et al., 2000).”

“…metabolite flux through oligodendrocytes can in turn be regulated by axonal activity: glutamate activation of NMDA receptors on oligodendrocytes increases their uptake of glucose, which may then be metabolized to lactate and released to axons for oxidative ATP production (Saab et al., 2016).”

“transformed into lactate which is further locally released from OLs toward axons through the monocarboxylate transporter MCT1 expressed at the inner tongue of myelin sheaths (Saab et al., 2016).”

“This led to a working model that oligodendroglial glutamate receptor signaling matches -with some temporal delay-the glycolytic flux and pyruvate/lactate export to axonal energy demands [46].”

“Activation of oligodendrocyte NMDA receptors upregulates oligodendrocyte glucose uptake, which has been proposed to alter their metabolic support of axons (Saab et al., 2016).”

“Myelin, produced by oligodendrocytes in the CNS, electrically insulates axons to facilitate saltatory conduction (Rushton, 1951; Cohen et al., 2020; Hartline and Colman, 2007) and also provides metabolic support (Fünfschilling et al., 2012; Lee et al., 2012; Saab et al., 2016).”

“Electrophysiological recordings were done as described [54, 69].”

“…(Bankston, Mandler, & Feng, 2013; Dougherty, Dreyfus, & Black, 2000; Jang, Gould, Xu, Kim, & Kim, 2019; Lee at al., 2012; Philips & Rothstein, 2017; Saab et al., 2016; Wilkins, Majed, Layfield, Compston, & Chandran, 2003) and to the regulation of axonal excitability through K+ buffering (Larson…”

“As noted throughout, OPCs have the ability to respond to neuronal activity, but recent studies have suggested that they might in turn also tune neuronal function by releasing secreted factors (Sakry et al. 2014) and neuromodulators to locally regulate the function of neuronal synapses.”

“As myelination has been established to function critically in providing axonal metabolic support (Lee et al., 2012b; Fünfschilling et al., 2012; Saab et al., 2016), myelination of PV+ interneurons could function by helping to optimize axonal energy utilization.”

“GLUT1 upregulation occurs within minutes and augments glucose import, stimulating lactate release [66].”

“This metabolic support must be carefully balanced with axonal energy needs to prevent local acidosis and damage 40,41 .”