The nuclear Cox10
gene encodes a haem A farnesyl transferase, essential for the assembly of COX, the terminal complex of the electron transport chain in mitochondria7, 8
. In COX10 mutants, COX is unstable and rapidly degraded. Cox10flox/flox
mice have been used to generate models of severe mitochondrial disease in muscle, brain and liver7, 8, 9, 10
. We crossed the Cox10flox/flox
that express Cre recombinase in Schwann cells and oligodendrocytes (with Cre expression beginning at the respective precursor stages in these cells)11, 12
. In the absence of functional COX, glial cells should fail to fully metabolize glucose, and should generate ATP mostly by glycolysis and produce lactate. Thus, demyelination and glial cell death would be key indicators for metabolic demands of glial cells, toxic effects of lactic acidosis, and metabolic coupling between neuronal and glial compartments in myelinated fibre tracts.
Crosses of Cox10flox/+*Cnp1Cre/+ and Cox10flox/flox*Cnp1+/+ mice led to fewer mutant pups (Cox10flox/flox*Cnp1Cre/+) than expected (11% compared with 25% expected, n = 466), suggesting prenatal death of some mice, probably owing to the known Cnp1Cre/+ expression in a subset of neural precursors (see later). We therefore generated other mutants by selectively targeting Schwann cells (using Dhh-Cre mice) and mature oligodendrocytes (using tamoxifen-inducible Plp1-CreERT2 mice), and found no evidence of embryonic lethality. In the following, data are fromCnp1Cre/+*Cox10flox/flox mutants (and Cnp1Cre/+*Cox10flox/+ controls), unless otherwise stated.
Quantitative polymerase chain reaction (qPCR) of genomic DNA from neural tissue (at postnatal day (P) 21) confirmed the loss of exon 6 and thus a functional Cox10
gene (). For the sciatic nerve we calculated that 67 ± 2% (mean ± s.e.m., n
= 4) of all cells were recombinant (assuming that both alleles recombine). Thus, most Schwann cells in peripheral nerves were successfully targeted. In the optic nerve and cervical spinal cord, we calculated that about 50% of cells were recombined at the Cox10
locus (), the estimated percentage of oligodendrocytes13
Genetic targeting of the mitochondrial COX complex in myelinating glial cells
Elimination of Cox10
is not immediately followed by the loss of respiration, but predicts the rapid functional ‘ageing’ of mitochondria, as the mitochondrial half-life is about 3 weeks in the brain14, 15
. Thus, postnatal myelination by oligodendrocytes will involve intact mitochondria (). By contrast, recombination of Cox10
in proliferating Schwann cells causes a more rapid ‘loss’ of intact mitochondria by dilution in the progeny (, right). Indeed, the absence of COX from many Schwann cells was obvious at P21 (). For comparison, immunostaining of axonal mitochondria was indistinguishable between mutants and controls. We also noted occasional spinal motor neurons lacking COX (not shown), a plausible contributor to embryonic lethality.
A severe neuropathy phenotype proved that gene targeting was efficient and disruptive for mitochondrial function. Both Cnp1Cre/+*Cox10flox/flox and Dhh-Cre*Cox10flox/flox mice exhibited a developmental defect, with reduced weight, tremors and hindlimb weakness, although mutants appeared otherwise healthy and well groomed. The disease progressed to hindlimb paralysis and muscle atrophy (Supplementary Fig. 1a), followed by forelimb paralysis at about 9 months, when mutants had to be euthanized.
Already at P21 attempts to compare sciatic nerve conduction velocities between controls (15.6 ± 0.7 m s−1; mean ± s.e.m.; n = 5) and mutant mice (n = 5) failed, indicating conduction blocks (). Only distal stimulation elicited a compound muscle action potential in mutant mice, but with 50% reduced amplitude (Supplementary Fig. 1b), indicating a combination of glial and axonal perturbations. The same functional blocks were seen in Dhh-Cre*Cox10flox/flox mice (not shown), confirming the specificity for Schwann cells.
Peripheral neuropathy caused by Cox10 mutant Schwann cells
By morphology and TdT-mediated dUTP nick end labelling (TUNEL) staining, we found no evidence for Schwann cell death as a possible cause of hypomyelination (not shown). In fact, the number of cell nuclei was increased (). Compared with controls, mutant nerves were thinner (), the absolute number of myelinated axons was reduced () and many medium-sized axons remained unmyelinated (Supplementary Fig. 1c, d). However, neither large nor small axons were significantly altered in number (Supplementary Fig. 2a), suggesting a primary dysmyelination at P21. By immunostaining for the activated monocyte/macrophage marker MAC3, reactive macrophages were found to not yet be a feature (not shown), confirming the developmental defect. At later stages, the number of myelinated axons was reduced (), and in 2-month-old mice fibre degeneration was already associated with secondary inflammation (Supplementary Fig. 2b). Notably, even at 9 months there was no major loss of Schwann cells, with 193 ± 25 per sciatic nerve section in mutants and 239 ± 17 in controls (mean ± s.e.m., n = 4).
Where myelin was present, g
-ratio analysis (the ratio between the diameter of the inner axon and the total outer diameter) showed a decrease in myelin thickness (0.589 ± 0.009 in controls; 0.612 ± 0.012 in mutants; mean ± s.d., n
= 3). By electron microscopy, many axons that appeared correctly sorted remained unmyelinated, despite a calibre >1.0 μm (). Remak cells failed to engulf single axons with cellular processes (). Thus, the axonal sorting defect in mice lacking the transcription factor TFAM1 (ref. 16
) is probably the consequence of perturbed COX activity in Schwann cells.
Mitochondria were visibly enlarged in mutant Schwann cells (), but not in associated axons (Supplementary Fig. 2c), reflecting a loss of the proton gradient and a failure of H+
. Compacted myelin, when present, had a normal ultrastructure and was stable ().
An intriguing aspect of altered energy metabolism in peripheral nerves was the increase of vascularization. Blood vessels covered a threefold larger area in cross-sections from mutant nerves (Supplementary Fig. 3a–c). Hypervascularization was associated with upregulation of Vegfamessenger RNA, but not of Hif1a or glucose transporters (Supplementary Fig. 3d), and is possibly a response to enhanced glycolysis.
Myelinated tracts were normally developed in brains and spinal cords of mice at 2 months of age (Supplementary Fig. 4), which is best explained by sufficient respiration of oligodendroglial mitochondria during myelination (). The fact that losing mitochondrial function requires several weeks was confirmed by inactivating Cox10
in postmitotic neurons (at P5–P10) usingCamKII-Cre
mice. As previously shown10
, these mice die prematurely with severe neurodegeneration, visualized by neuroinflammation and loss of hippocampal neurons at 4 months of age (Supplementary Fig. 5).
Surprisingly, even at 9 months of age when Cnp1Cre/+*Cox10flox/flox mice had to be killed because of peripheral neuropathy, we detected no signs of white matter pathology, demyelination or oligodendroglial pathology (), although the absence of COX activity from oligodendrocyte lineage cells was obvious by sequential COX and succinate dehydrogenase (SDH, also known as mitochondrial complex II) histochemistry ( and Supplementary Fig. 6). Mature oligodendrocytes (CC1+) instead showed an abnormal (SDH+) mitochondrial expansion, but were COX− (). To extend the observational time window, we induced recombination by tamoxifen inPlp1-CreERT2*Cox10flox/flox 1-month-old mice. Histological analysis at age 14 months (the latest time-point tested) also failed to show any signs of demyelination or neurodegeneration (Supplementary Fig. 7a–c). Furthermore, magnetic resonance imaging (MRI) showed that there was no difference in ventricular volume between mutants (8.1 ± 1.1 mm3 (mean ± s.d.)) and controls (8.4 ± 1.4 mm3) at 6–7 months of age (Supplementary Fig. 7d, e).
Oligodendroglial survival, myelin preservation and white matter integrity inCnp1Cre/+*Cox10flox/flox mice
Theoretically, white matter integrity could be preserved when mutant oligodendrocytes are replaced by newly generated oligodendrocytes that remyelinate. These cells might again survive for several weeks after recombination of Cox10
. We think this is unlikely for several reasons. By electron microscopy and g
-ratio analysis, central nervous system (CNS) myelin thickness in adult mutants was in the normal range ( and Supplementary Fig. 8), unlike remyelinated axons that are hypomyelinated18
. Moreover, we failed to observe apoptotic cells in the white matter tracts of adultCox10
mutants (Supplementary Fig. 9a), and the administration of BrdU did not label more oligodendrocyte precursor cells in mutants than in age-matched controls (Supplementary Fig. 9b). Furthermore, staining for astrocytes, microglia and T cells (Supplementary Fig. 10) gave no indication of low-grade inflammation, a very sensitive response to degenerative processes in white matter tracts. We conclude that once myelination has occurred, reduced mitochondrial functions do not perturb oligodendrocyte survival, myelin maintenance or axonal integrity.
We proposed that oligodendrocytes survive by enhanced glycolysis, and predicted to see in vivo an increase in the brain lactate concentration using localized proton magnetic resonance spectroscopy (MRS). As expected, Cox10 mutants showed a more strongly increased lactate resonance (compared with controls) in both cortex and white matter. Notably, accumulated brain lactate was detectable only under isoflurane anaesthesia (). When quantified in adult mutants (), we determined >4 mM lactate in the cortex and >6 mM in the corpus callosum (compared with <2 mM lactate in the cortex and <4 mM in the white matter of wild-type mice). At the end of anaesthesia, these lactate concentrations fell within minutes to very low levels (). The latter finding is compatible with a model in which oligodendroglial release of lactate — the necessary by-product of aerobic glycolysis — is followed by its rapid use in other cellular compartments. It is demonstrated in vitro that glial lactate is efficiently metabolized by myelinated axons (Supplementary Fig. 11). In vivo, lactate levels never reached MRS detectability under physiological conditions, unless challenged by isoflurane anaesthesia. Of note, the levels of N-acetylaspartate (a marker of viable neurons and axons), choline-containing compounds (indicators of membrane turnover) and myo-inositol (an osmolyte and glial membrane constituent) further support our conclusion that neurodegeneration and abnormal oligodendrocyte turnover are not a feature (Supplementary Fig. 12).
Rapid use of lactate shown by proton MRS
COX10-deficient cells in culture are practically respiration-deficient19
. Because we combined well-established lines of floxed mice and Cre transgenic tools, the reported phenotype is probably caused by the loss of mitochondrial ATP generation. In the periphery, the mitotic expansion of (mutant) Schwann cell precursors rapidly dilutes functional mitochondria, and hypomyelination demonstrates that myelination is an energy-consuming process. We also note that many patients with mitochondrial disorders have signs of neuropathy with hypomyelination. By contrast, unperturbed CNS myelination is best explained by sufficient residual function of mitochondria in mature oligodendrocytes after recombination (). We note that germline mutations of humanCOX10
and other COX assembly factors, although ultimately lethal, have been associated with CNS dysmyelination (leukodystrophy)8
Most intriguing is the mild post-myelination CNS phenotype of adult Cox10
mutant mice, in which oligodendrocytes maintain myelin and their ensheathed axons. That mature oligodendrocytes apparently survive by aerobic glycolysis similar to tumour cells (known as the Warburg effect) was unexpected. In fact, oligodendrocytes in vitro
are sensitive to the COX inhibitor azide5
. However, these cells correspond to ‘pre-myelinating’ oligodendrocytes in vivo
, and it seems that metabolic properties of oligodendrocyte lineage cells change during maturation. After myelination, oligodendrocytes may tolerate a partly glycolytic metabolism that we have experimentally augmented in Cox10
mutants. We note that mature wild-type oligodendrocytes have lower COX activity than oligodendrocyte precursor cells (not shown), and that white matter has relatively high glycolytic activity20
. Also, in our control mice absolute lactate levels (as a surrogate marker for aerobic glycolysis) were higher in white than grey matter ().
Enhanced glycolysis in oligodendrocytes increases the production of pyruvate, lactate and acetyl-coenzyme A (CoA), the latter promoting fatty acid synthesis during myelination. Because energy-deprived myelinated axons can rapidly use pyruvate and lactate as an alternative energy source6,21
(Supplementary Fig. 11), we suggest a model in which an increased rate of oligodendroglial glycolysis can supply glycolysis products to support axonal energy needs ().
Our observation that lactate levels in mutant brains are visibly increased compared with controls, but only under isoflurane anaesthesia, suggests that lactate is not cleared by drainage, but is locally metabolized. Isoflurane anaesthesia does not decrease CNS blood flow. The theoretical possibility that increased lactate peaks are caused by mutant oligodendrocytes that fail to metabolize neuronal lactate seems remote (note the similar lactate increases in grey matter, where neurons are associated with respiration-competent astrocytes22
). Increased lactate concentrations probably originate from Cox10
mutant oligodendrocytes that rely on increased glycolysis for survival. Even under normal conditions, lactate exported from oligodendrocytes would be rapidly metabolized and therefore not be detected by MRS.
By in situ
and immunoelectron microscopy24
, the monocarboxylate transporter MCT1 (also known as SLC16A1) is expressed in white matter, and is even present in myelin24
, whereas MCT2 localizes to myelinated axons. This strongly suggests that lactate generated in oligodendrocytes is released and reaches the axonal compartment ().
We assume that oligodendrocytes can oxidize glycolysis-derived NADH+
when mitochondria are intact (using a NADH-glycerolphosphate shuttle that is oligodendrocyte-specific25
). Thus, normal oligodendrocytes may not even reduce pyruvate, which passes equally well through MCT1 and MCT2. Such a model of oligodendrocytes, yielding glycolysis products to axons when energy deprived4
, does not contradict the presumed role of lactate in the support of myelination during development24
, that is, when the import of metabolites is rate-limiting for lipid synthesis26
, specifically for oligodendrocytes in culture24
Our model of axon–oligodendrocyte metabolic coupling is reminiscent of the astroglial ‘lactate shuttle’, which is thought to support the energy metabolism of glutamatergic synapses in the cortex27, 28
. For oligodendrocyte lineage cells, the inferred developmental ‘switch’ of metabolism even allows them to survive by aerobic glycolysis. The underlying mechanisms deserve further attention, as they may be relevant for the survival of glioma cells and the Warburg effect.