Distribution of monocarboxylate transporters in the peripheral nervous system suggests putative roles in lactate shuttling and myelination

Abstract

Lactate, a product of glycolysis, has been shown to play a key role in the metabolic support of neurons/axons in the CNS by both astrocytes and oligodendrocytes through monocarboxylate transporters (MCTs). Despite such importance in the CNS, little is known about MCT expression and lactate function in the PNS. Here we show that mouse MCT1, MCT2, and MCT4 are expressed in the PNS. While DRG neurons express MCT1, myelinating Schwann cells (SCs) coexpress MCT1 and MCT4 in a domain-specific fashion, mainly in regions of noncompact myelin. Interestingly, SC-specific downregulation of MCT1 expression in rat neuron/SC cocultures led to increased myelination, while its downregulation in neurons resulted in a decreased amount of neurofilament. Finally, pure rat SCs grown in the presence of lactate exhibited an increase in the level of expression of the main myelin regulator gene Krox20/Egr2 and the myelin gene P0. These data indicate that lactate homeostasis participates in the regulation of the SC myelination program and reveal that similar to CNS, PNS axon-glial metabolic interactions are most likely mediated by MCTs.

Keywords: Schwann cells; lactate; monocarboxylate transporters; peripheral nervous system.

Figure 1.

The expression of MCT1, MCT2, and MCT4 in the PNS. A, One percent agarose gel with PCR products from reverse transcription. All three transporters were detected in purified mouse SCs, and MCT1 and MCT2 were also detectable in purified mouse DRG neurons (mouse neu). Non-retrotranscripted RNA was used as a negative control (C−). B, The expression of MCT1, MCT2, and MCT4 was analyzed by quantitative PCR, using ubiquitin-conjugating enzyme E2 as a reference. When possible (P28 and P56), the perineurium was removed from the sample and only the endoneurium was studied. Each time point corresponds to measurements performed on samples isolated from two animals and performed in triplicates. Error bars indicate SE.

Figure 2.

Localization of MCT1 in the PNS. A, The MCT1 blocking peptide abrogates the MCT1 staining on teased fibers. Both images are shown with the same intensity level. B, Immunostaining on cross section from adult mouse sciatic nerve shows the presence of MCT1 in both perineurium (p) and endoneurium (e). C, Immunostaining on cross section from adult mouse DRG revealed that MCT1 is particularly enriched in the “capsule” (arrowheads) and at the surface of soma of a subset of sensory neurons. D, MCT1 is strongly present at the SLI, colocalizing with phalloidin (arrowheads). E, MCT1 colocalizes with Caspr indicating its presence in paranodal regions (arrowheads). Inserts show a magnification of the nodal and paranodal region. Scale bars: A, C–E, 20 μm; B, 50 μm.

Figure 3.

Localization of MCT4 in the PNS. A, Cultured rat SCs were lipofected with scrambled or MCT4 siRNA. MCT4 localized to the perinuclear cytoplasm (arrowheads), and the signal was abrogated when MCT4 expression was knocked down, demonstrating both the presence of the transporter and the specificity of the antibody. B, Immunostaining on cross section from adult mouse sciatic nerve revealed the presence of MCT4 in the endoneurium (e). C, D, MCT4 is expressed in myelinated teased fibers in a thin line and a mesh-like shape (arrowheads), suggesting localization in the outer cytoplasmic mesaxonal line and Cajal bands. MCT4 expression is also visible in perinuclear cytoplasm (arrow). E, Cross section of sciatic nerve reveals the presence of MCT4 in the perinuclear cytoplasm (arrows) and in the most abaxonal part (arrowheads) of myelinated SCs. F, MCT4 appears as a discontinuous patchy pattern at the most abaxonal part of the fiber (arrowheads), above the myelin sheath stained by MBP. Scale bars: A, C, D, 20 μm; B, 50 μm; E, F, 10 μm. P, perineurium.

Figure 4.

Downregulation of MCT1 in vitro leads to cell type-specific phenotypes and modulates the transcription of myelin-related markers in purified SCs. Rat DRG neurons (Neu) and SCs were infected with lentiviruses containing shRNA for MCT1 (KD) or scramble (cont) and selected with puromycin. The neurons and SCs were cocultured for 2 weeks in myelinating conditions. All experiments were performed in triplicates. All values refer to the scramble (controls), normalized as 1 or 100. A, On average, 87 and 75% of MCT1 mRNA depletion was achieved in purified SCs and DRG neurons, respectively. B, When MCT1 KD SCs were cocultured with control neurons, a significant increase in Krox20 and P0 protein level was revealed (Krox20 292.2 ± 43.63, p = 0.023; P0 3082.34 ± 875.49, p=0.038). Cocultures in which only neurons were depleted for MCT1 showed a significant reduction for NF200 (55.69 ± 1.5, p < 0.001) without significant alteration in the myelin markers. The histogram illustrates the quantification of protein expression from Western blots, normalized to actin. C, Rat SCs were infected with lentivirus carrying scrambled or MCT1 shRNAs and selected in SC proliferative media in the presence of puromycin. Then, cells were switched to myelinating media containing 10% FBS and ascorbic acid, and the mRNA was extracted after 2 d. The experiments were performed in triplicates. With >70% of MCT1 depletion (0.27 ± 0.03, p < 0.001), mRNAs for myelin markers were increased (Krox20 2.48 ± 0.37, p = 0.017; P0 1.28 ± 0.03, p < 0.01). D, Rat SCs were seeded in poly-l-lysine-coated dishes and cultured in myelinating media containing 10% FBS and ascorbic acid for 1 d. Then 2 mm of l-lactate was added to the myelinating media and cells were harvested for mRNA extraction at indicated time points. The experiment was performed in triplicate and the values were referred to the control. Twenty-four hours after lactate addition, transcription of Krox20 was significantly increased (1.32 ± 0.07 p = 0.022), and this increase was enhanced at 48 h (2.27 ± 0.13, p = 0.005). The expression of P0 was also transiently increased 24 h after addition of lactate (1.15 ± 0.05, p = 0.049), returning to control levels at 48 h (1.03 ± 0.03, p = 0.182).