Regulation of oligodendrocyte development and myelination by glucose and lactate - PubMed (original) (raw)
Regulation of oligodendrocyte development and myelination by glucose and lactate
Johanne E Rinholm et al. J Neurosci. 2011.
Abstract
In the gray matter of the brain, astrocytes have been suggested to export lactate (derived from glucose or glycogen) to neurons to power their mitochondria. In the white matter, lactate can support axon function in conditions of energy deprivation, but it is not known whether lactate acts by preserving energy levels in axons or in oligodendrocytes, the myelinating processes of which are damaged rapidly in low energy conditions. Studies of cultured cells suggest that oligodendrocytes are the cell type in the brain that consumes lactate at the highest rate, in part to produce membrane lipids presumably for myelin. Here, we use pH imaging to show that oligodendrocytes in the white matter of the rat cerebellum and corpus callosum take up lactate via monocarboxylate transporters (MCTs), which we identify as MCT1 by confocal immunofluorescence and electron microscopy. Using cultured slices of developing cerebral cortex from mice in which oligodendrocyte lineage cells express GFP (green fluorescent protein) under the control of the Sox10 promoter, we show that a low glucose concentration reduces the number of oligodendrocyte lineage cells and myelination. Myelination is rescued when exogenous l-lactate is supplied. Thus, lactate can support oligodendrocyte development and myelination. In CNS diseases involving energy deprivation at times of myelination or remyelination, such as periventricular leukomalacia leading to cerebral palsy, stroke, and secondary ischemia after spinal cord injury, lactate transporters in oligodendrocytes may play an important role in minimizing the inhibition of myelination that occurs.
Figures
Figure 1.
Lactate uptake in corpus callosum generates an intracellular pH change. Cell bodies and processes aligned with axons, which are presumed to be oligodendrocyte processes, in corpus callosum loaded with BCECF-AM, respond to lactate with a reduction in fluorescence intensity excited at 488 nm, but no change in fluorescence excited at 458 nm, indicating acidification. A, Confocal image from corpus callosum showing the regions measured in B–E. R1 and R2 are processes aligned with axons, whereas R3 is a cell body (this soma was chosen to have a fluorescence level that does not saturate the detector). B, The fluorescence response to 10 m
m
lactate in region R1 at excitation wavelengths 488 and 458 nm. C, The fluorescence response (F) to lactate in region R1 shown as the ratio _F_488/_F_458. D, The fluorescence response to lactate in region R2 (_F_488/_F_458). E, The fluorescence response to lactate in region R3 (_F_488/_F_458).
Figure 2.
The MCT inhibitors phloretin, 4-CIN, and NPPB reduced the pH response to lactate in the corpus callosum. A, Three successive pH responses (measured from regions encompassing numerous BCECF-loaded processes and somata in the corpus callosum) to 10 m
m
lactate. B, As in A, but with 100 μ
m
phloretin present during the second response. C, The mean responses to lactate ± SEM with and without phloretin in six slices for each condition. D, The mean responses to lactate with and without 100 μ
m
NPPB (n = 4 and 6 slices, respectively). E, The mean responses to lactate with and without 1 m
m
4-CIN (n = 5 and 6 slices, respectively). F, Comparison of the second response to lactate (lac 2) without drug (control), with the mean responses in phloretin, 4-CIN, or NPPB. All the inhibitors reduced the lactate response. Values of p are from two-tailed t tests comparing the second responses in control and drug conditions as in C–E.
Figure 3.
Lactate uptake in cerebellar white matter and in whole-cell patch-clamped oligodendrocytes. A, Confocal image from the cerebellum showing white matter with granule cells on each side. B, Three successive pH responses to 10 m
m
lactate measured in the white matter. C, As in B, but with 100 μ
m
phloretin present during the second response. D, The mean responses to lactate with and without phloretin in six slices with and four slices without phloretin. Error bars indicate SEM. E, Whole-cell patch-clamped oligodendrocyte in the cerebellar white matter filled with BCECF from the pipette. Left, Fluorescence image of the oligodendrocyte. The soma and the parts of the processes that were monitored are indicated (black and white rectangles, respectively). Middle, The fluorescence response to 10 m
m
lactate in the soma of the cell. Right, The fluorescence response to lactate in the indicated processes. In both areas, lactate evokes a clear acidification, superimposed on some baseline drift.
Figure 4.
MCT1 is expressed in myelin. A–C, Single confocal images from the cerebellar white matter labeled with antibodies to MBP, MCT1 (A), MCT2 (B), and MCT4 (C). The insets are higher magnification images from each slice. Scale bars: insets, 1 μm. A, MCT1 (green) strongly labels blood vessels, but some labeling is also seen in the myelin (MBP, red). Colocalization is seen as yellow spots in the overlay and is indicated by white arrowheads. B, MCT2 (green) apparently colocalizes with MBP (red). Colocalization is seen as yellow spots in the overlay and is indicated by white arrowheads. However, EM studies (see F and main text) show that the MCT2 is actually in axons. C, MCT4 (green) labels processes presumed to be astrocytic but does not colocalize with MBP (red). D, Electron micrographs of a myelinated axon (left) and part of an endothelial cell surrounding a blood vessel lumen (right) in the cerebellar white matter labeled with antibodies to MCT1 coupled to gold particles (15 nm black dots indicated by red arrowheads). Mito, Mitochondrion; astro, astrocyte; endothel, endothelial cell. E, Density of gold particles labeling MCT1 in white matter axons, endothelial cells (Endo), and myelin. F, Electron micrographs of a myelinated axon in the cerebellar white matter (left) and a parallel fiber–Purkinje cell synapse in the cerebellar molecular layer (right) labeled with antibodies to MCT2 coupled to gold particles (10 nm black dots indicated by red arrowheads). G, Density of gold particles labeling MCT2 in axons, postsynaptic density (PSD) from parallel fiber–Purkinje cell synapses, and myelin. Error bars indicate SEM.
Figure 5.
A decrease in glucose concentration inhibits oligodendrocyte development and myelination in cultured cortical brain slices. A, Images of the cerebral cortex in cultured brain slices from mice in which oligodendrocyte lineage cells express GFP under control of the Sox10 promoter. The slices were cultured in solution containing (from left to right) 41.5, 10.5, 5.5, 2.9, and 1.4 m
m
glucose and immunolabeled for MBP (red) and NF (blue). Sox10-GFP is green. B–D, Normalized fluorescence intensity (see Materials and Methods) for MBP (B), NF (C), and Sox10-GFP (D), pooled from four separate experiments, using a total of 22, 10, 23, 24, and 9 slices for 41.5, 10.5, 5.5, 2.9, and 1.4 m
m
glucose, respectively. E, F, Labeling intensity ratio for MBP/NF (E) and MBP/Sox10-GFP (F) in 41.5, 10.5, 5.5, 2.9, and 1.4 m
m
glucose. G, Sox10-GFP fluorescence (arbitrary units) versus number of Sox10-GFP cells (counted using ImageJ), demonstrating a linear relationship between the two in 30 slices cultured in 1.4–41.5 m
m
glucose (each point is one slice). Error bars indicate SEM.
Figure 6.
The effect of
l
- and
d
-lactate on oligodendrocyte development and myelination in 41.5 and 2.9 m
m
glucose. A–C, Normalized fluorescence for MBP (A), NF (B), and Sox10-GFP (C), pooled from two experiments using a total of 15 slices for control and
d
-lactate, and 10 slices for
l
-lactate. D, E, Labeling intensity ratio of MBP/NF (D) and MBP/ Sox10-GFP (E) in the same slices as A–C. F, Images of the cerebral cortex in cultured brain slices from mice in which oligodendrocyte lineage cells express GFP under control of the Sox10 promoter. The slices were cultured in solution containing (from left to right) 41.5 m
m
glucose, 2.9 m
m
glucose, 2.9 m
m
glucose with 20 m
m d
-lactate, or 2.9 m
m
glucose with 20 m
m l
-lactate. Slices were immunolabeled for MBP (red) and NF (blue). Sox10-GFP is green. G–I, Normalized fluorescence for MBP (G), NF (H), and Sox10-GFP (I), pooled from two separate experiments, using a total of 11 slices for 41.5 m
m
glucose, and 13 slices for each condition in 2.9 m
m
glucose. J, K, Labeling intensity ratio of MBP/NF200 (J) and MBP/Sox10-GFP (K) in the same slices as G–I. Error bars indicate SEM.
Figure 7.
Schematic illustration of glucose and lactate fluxes between blood vessels, astrocytes, oligodendrocytes, and neurons. Glucose is transported across the blood–brain barrier through GLUT1 expressed on endothelial cells and can be taken up by astrocytes through GLUT1 on astrocytic endfoot membranes. Glucose may also be taken up by oligodendrocytes (through GLUT1) and neurons (through GLUT3). Astrocytes, which can store glycogen, can export lactate through MCT4. The lactate may be taken up by oligodendrocytes through MCT1, or by neurons through MCT2. At times of high blood lactate concentration, astrocytes and oligodendrocytes, which both are in direct contact with blood vessels, may take up lactate from the blood. The lactate is converted to pyruvate, which can be used for ATP production and in oligodendrocytes may be particularly important to produce lipids for myelination.
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