Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism

Abstract

Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Figure 1.. NMDA Stimulates GLUT1 Surface Expression and Glucose Uptake by Cultured Oligodendrocytes

(A) Oligodendrocyte immunostained for GLUT1 and GalC. NMDA receptor stimulation mobilizes GLUT1 and increases its cell surface expression, which is blocked by D-AP5. Scale bar, 20 μm. (B) GLUT1 and MCT1 immunoblots following cell surface biotinylation of immunopanned NMDA-treated oligodendrocytes and controls. (C) Quantification of the experiments giving the ratio of GLUT1:GalC in a stained area of n = 3 experiments and 20–24 cells per condition (*p Finteraction(251, 7,109) = 1.96, p < 0.0001, two-way ANOVA). (G) Quantification of calibrated FRET signals of NMDA-stimulated oligodendrocytes (26–28 min), compared to baseline and cells also receiving 7CKA and D-AP5 (***p 13C, 1H-HSQC spectra at 700 MHz corresponding to proton resonance from the culture medium of immunopanned oligodendrocytes 30 min after exposure to NMDA/Gly (top) and controls (bottom). Boxed: lactate signals. Quantification by comparison of the peak volume of lactate methyl group to the internal standard DSS. (J) Increased lactate following NMDA/Gly treatment, depicted by overlaying the 1D-1H slices of spectra in the lactate methyl signal range. (K) Lactate release after NMDA receptor stimulation increased to 124% ± 4% (n = 4 paired NMR experiments, p = 0.023 paired t test).

Figure 2.. Oligodendroglial NMDA Receptor Mutants with Reduced GLUT1 Incorporation into Myelin

(A) Immunostaining of NR1 in optic nerve cross sections at age P80 from control and NR1 cKO mice (left panel). Higher magnifications (middle and right panels) reveal that NR1 (green) is absent from mutants and overlaps with MBP (red) in control mice. Scale bars, 20 μm (left) and 1 μm. See also Figure S2A. (B) Quantitation of NR1 staining intensity from whole optic nerve sections (n = 3; p = 0.019, Student’s t test). (C) Cre-mediated recombination of genomic DNA in individual optic nerves of each genotype as quantified by qPCR. Depicted is the relative abundance of the floxed NR1 allele after recombination (normalized to the abundance in NR1fl/fl littermate controls, defined as 100% at different ages). In NR1 mutant nerves, the abundance of NR1 flox copies was determined at ages P5 (98% ± 22% flox copies remaining; n = 5 versus 4, p = 0.939), age P10 (69% ± 7%; n = 4, p = 0.0119), age P16 (60% ± 4%; n = 4 versus 3, p = 0.0024), and P96 (38% ± 2%; n = 4 versus 9, p < 0.0001). (D) Western blot analysis of NR1 expression in individual nerves from mutant and control mice at age P75. Quantification revealed a reduction by 61% ± 12% in NR1 cKO nerves (n = 3, p = 0.0079). GAPDH, loading control. (E) By western blotting, GLUT1 is reduced in purified myelin of NR1 mutants (Sirt2, loading control). (F) Quantification of E (n = 3; p = 0.008, Student’s t test). (G) Localization of GLUT1 by immunogold labeling. In optic nerve cross sections, GLUT1 was detected in myelin sheaths, the outer tongue, and paranodal loops. Scale bar, 200 nm. Gold particles, red arrows. (H) Reduced abundance of immunogold labeled GLUT1 in myelin of NR1 mutant optic nerves when compared to littermate controls (10–12 randomly taken images per animal, n = 3; p = 0.0025, Student’s t test). (I) By immunogold labeling, MCT1 is associated with adaxonal (outer tongue) and abaxonal (inner tongue) myelinic channels, without difference between mutants and controls. Scale bar, 100 nm.

Figure 3.. Myelination in the Absence of Oligodendroglial NMDA Receptors In Vivo

(A–C) High-pressure freezing (HPF) electron microscopy of the developing optic nerve. Overview of optic nerve cross sections from control and NR1 mutant mice at P10 (A), P20 (B), and P70 (C). At early and late stages, NR1 mutant nerves are indistinguishable from controls. A minor hypomyelination is apparent at P20. Scale bars, 0.5 μm (A and B) and 0.2 μm (C). (D) Electron microscopy of conventionally fixed optic nerves from mutant and controls, with unmyelinated (U), ensheathed (E), and myelinated (M) axons. Scale bar, 1 μm. (E–I) Axon size distribution and myelin sheath thickness (g-ratio) at P18 and P70. (E) Diameter spectrum of myelinated axons with relatively more myelinated small caliber axons in control nerves than in NR1 cKO at P18 (***p

Figure 4.. Axonal Energy Metabolism Regulated by NMDA Receptor and GLUT1-Dependent Lactate Export from Myelinating Oligodendrocytes

(A) Top: scheme of recording compound action potentials (CAPs) from acutely isolated optic nerves. After 1 hr, nerves were subjected to 60 min oxygen glucose deprivation (OGD) followed by reperfusion with ACSF containing 10 mM glucose. Bottom: optic nerve CAP areas (i.e., area underneath CAPs as shown in C) normalized to baseline. Note the rapid decline of nerve conduction and the incomplete recovery after reperfusion, which is more pronounced in NR1 mutants (red) compared to controls (black). (B) Averaged optic nerve CAPs during baseline, OGD, and recovery phase in control (top) and NR1 cKO (bottom). (C) Quantification of data in (A) with reduced functional recovery after OGD in mutants (n = 12) versus controls (n = 11, p = 0.0046, Student’s t test). (D) With 20 mM lactate, the functional recovery after OGD was the same in NR1 mutants (n = 6) and controls (n = 8). (E) Axonal recovery at higher temporal resolution, comparing 10 mM glucose (glc) and 20 mM lactate (lac). Depicted are fitting curves (lines, Boltzmann fit) for the average CAP area over time (p

Figure 5.. Requirement of Oligodendroglial NMDA Receptors for High-Frequency Conduction

(A) Scheme of stimulating DRG axons of lumbar segment L4 and recording from fasciculus gracilis at L1 (inset: intact myelination of spinal cord in mutants and controls, Gallya’s stain). (B) Representative CAPs (averaged) in control (top) and mutants at age 4–6 months. Dotted line: peak-to-peak amplitude used to analyze firing strength of fastest axon groups. Conduction delays were unchanged. (C) Normal axonal excitability in mutants measured at increasing stimulus intensities with peak-to-peak amplitude normalized to maximal readings. (D) After 10 min at 0.1 Hz, stimulation frequency increased to 10 Hz (medium frequency stimulation, MFS), showing no difference between mutants (n = 6) and controls (n = 3). (E) 100 Hz (high frequency stimulation, HFS) caused a decrease of firing strength (within seconds) that was significantly faster and stronger in NR1 mutants (n = 7) than controls (n = 5). Left inset: higher temporal resolution for indicated region, Finteraction (119, 1,190) = 3.07, p < 0.0001, two-way ANOVA). Right inset: slower recovery of CAPs at the end of HFS, monitored at 0.1 Hz (τ calculated from normalized exponential fits, p = 0.0084, Student’s t test).

Figure 6.. Late-Onset Neuroinflammation and Axonopathy in NR1 cKO Mice

(A) Indirect signs of neurodegeneration in white matter tracts at 1 year of age and overview (left) of sagittal cerebellum-spinal cord section from NR1 cKO mice. Signs of local inflammation (Mac3 immunostaining) were more obvious in the ventral white matter (vWM) compared to spinal cord gray matter (GM). Higher magnification (region marked by a red rectangle) reveals activated microglia (Mac3). Scale bar, 20 μm. See also Figure S7. (B) Neuroinflammation in the ventral white matter of NR1 cKO mice confirmed by increased density of cell nuclei (p = 0.029, n = 4, Student’s t test). In adjacent GM, nuclear densities are unaltered. (C) Quantification of the Mac3+ immunostained area (microgliosis) in vWM of NR1 mutant mice (p = 0.0004, n = 4, Student’s t test) (D) Quantification of the GFAP+ area (astrogliosis) in vWM of NR1 mutant mice (p = 0.013, Student’s t test). (E and F) By electron microscopy of ventral cervical spinal cord cross sections (E), ultrastructural features of axonal pathology and degeneration were more frequent in NR1 mutant mice compared to controls (F) (n = 4–5 mice with 12–14 randomly taken images, covering 530 μm2 each, p = 0.0014, Student’s t test). Scale bar, 2 μm. In (E): A, axonal degeneration; B, blebbing membranes; D, delamination; N, normal myelin. (G) Motor deficits of NR1 cKO mutants at 10–11 months of age, demonstrated by Rotarod testing on three consecutive days (repeated the following month). The latency to fall is decreased in NR1 mutant mice (red line) compared to littermate controls (Fgenotype (1, 17) = 4.95, p = 0.040, n = 9–10, two-way ANOVA). (H) At age 19 months, NR1 cKO mice (n = 11) display significant neurological deficits compared to controls (n = 8, p + show widespread signs of neuroinflammation in white matter tracts; corpus callosum and fimbria are magnified (right panel). (J) Quantification of Mac3+ immunostained area in NR1 mutants compared to littermate controls and age-matched Cnp1Cre/+ mice (n = 4–6 mice, p < 0.001, linear mixed effects analysis with post hoc Tukey correction for multiple comparisons). (K) Axonopathy in white matter tracts revealed by APP immunolabeling (arrow heads) in brain sections of 19-month-old NR1 cKO mice; corpus callosum, fimbria, and subventricular white matter are shown. (L) Quantification of APP spheroids in NR1 mutants in comparison to littermate controls and age-matched Cnp1Cre/+ mice (n = 4–6 mice; control versus NR1 cKO p < 0.001 and Cnp1Cre/+ versus NR1 cKO p = 0.03, linear mixed effects analysis with post hoc Tukey correction for multiple comparisons).

Figure 7.. Schematic Depiction of Oligodendroglial NMDA Receptor Signaling

Working model in which axonal electrical activity in developing white matter tracts constitutes a glutamatergic signal for the surrounding OPC/oligodendrocytes/myelin compartments (1). After myelination, NMDA receptors associated with the internodal/paranodal membrane respond to axonal glutamate release as a surrogate marker for increased axonal electrical activity and energy needs, causing (2) the incorporation of additional glucose transporters into oligodendrocytes and myelin and the adaptation of glucose uptake (feed-forward regulation). Glycolysis products (3) are initially used for ATP and lipid synthesis (4). Later, mature oligodendrocytes release lactate (or pyruvate) to fuel the axonal compartment (5) for mitochondrial ATP production (6). Regulation of oligodendroglial glucose uptake by axonal energy needs could help prevent abnormal accumulation of lactate. The possible effect of glutamate on glucose transporters on astrocytes is not shown.