Fig 2: Hypothetical model integrating the role of oligodendroglial MCT2 in the context of previous findings on white matter metabolism.A In control white matter, glucose (Glu; light blue circles) and monocarboxylates (ketone bodies (KBs), lactate (Lact), acetate (Ac), pyruvate (Pyr); brown circles) enter parenchyma via transporters expressed by endothelial cells lining the blood vessels (BV). Glucose is imported by astrocytes, lactate is generated and shuttled outside via MCT416. Astrocytes also metabolize fatty acids to KBs and export these via MCT493. Lactate94 and KBs can also be transferred to oligodendrocytes via connexin channels. MCT2 expressed by oligodendrocytes, given its high affinity, is predicted to efficiently import monocarboxylates from the extracellular space. Among these, KB, pyruvate and acetate can be metabolized to ATP and/or used as precursors for lipid synthesis. Lactate is likely not metabolized by oligodendrocytes because of low LDH expression25 but may be shuttled via myelinic channels and MCT1 on myelin to axons that transport it via MCT2. Oligodendrocytes also import glucose via Glut1, connexin hemichannels, or gap junctions from astrocytes94. Glucose can be directly shuttled to the axons or metabolized to pyruvate, which may then be shuttled to the axons25. B MCT2 deletion in oligodendrocytes is expected to significantly diminish monocarboxylate import under standard conditions, thus decreasing both the availability of carbon molecules and ATP for lipid synthesis. This leads to metabolic deficit that impairs myelin maintenance and likely compromises axonal metabolic support, resulting in axonal LDHA upregulation and damage. C Ketogenic diet increases KB entry to the CNS via endothelial MCT1. Moreover, it induces metabolic reprogramming of astrocytes allowing these to import KB via increased expression of MCT163. KB could then be delivered to oligodendrocytes via connexin channels. High extracellular KB concentrations might also allow lower affinity transporters such as MCT1, to import KB into oligodendrocytes and sustain lipid synthesis and axonal support. Moreover, ketones can be directly imported and metabolized by neurons/axons63.

Fig 3: Reduced expression of 2 major enzymes that regulate fatty acid synthesis in AAV-MCT2KO oligodendrocytes.A Schematic representation of fatty acid synthase (FASN) and acyl-coenzyme A synthetase short-chain family member 2 (ACSS2) involvement in fatty acid synthesis. ACSS2 regulates lipid synthesis via two distinct, not mutually exclusive mechanism. First, ACSS2 catalyzes the conversion of acetate (that can be imported via MCT2) to acetyl co-A. Acetyl Co-A then can be used as a substrate for, on one hand, palmitate synthesis by FASN (1st step of fatty acid synthesis), or on the other hand, for histone acetylation which enhances the expression of the genes that regulate lipid synthesis, including the gene encoding FASN. B Co-immunolabelling for GFP in green and FASN in gray. Yellow arrows indicate GFP + FASN+ cells, purple arrows indicate GFP + FASN- cells. C Quantification of the percentage of GFP+ cell expressing FASN at 3 weeks and 6 weeks. At 3 weeks (***) p = 0.0002, two-tailed Student’s unpaired t-test. Mean ± SEM. n = 5 for AAV-controls and n = 7 for AAV-MCT2KO. At 6 weeks, (**), p = 0.0040, two-tailed Student’s unpaired t-test. Mean ± SEM. n = 3 for AAV-controls and n = 5 for AAV-MCT2KO. D Co-immunolabelling for GFP in green and ACSS2 in red. White arrows indicate GFP + ACSS2+ cells and are more numerous in AAV-controls. E Quantification of the percentage of GFP+ cells expressing ACSS2 shows a significant decrease in AAV-MCT2KO at 3 weeks, (*) p = 0.0440, two-tailed Student unpaired t-test and at 6 weeks (**) p = 0.0070, two-tailed Student unpaired t-test. Mean ± SEM. n = 5 and 3 in AAV-controls at 3 and 6 weeks respectively and n = 4 and N = 5 in AAV-MCT2KO at 3 and 6 weeks, respectively. All scale bars = 20 µm.

Fig 4: Changes in MCT2 expression by oligodendroglia in patients with progressive MS.A SOX10+cell numbers in MS NAWM vs controls. Mean ± SEM. B Lines represent different patients; in 2/4 patients, SOX10+numbers increase perilesionally, 1/4 shows no change, and in 2/4 SOX10+ numbers decrease. Friedman test P = 0.0417. A significant downregulation of SOX10 was observed in the lesion core compared to the rim (perilesion), *p = 0.04, Dunn’s multiple comparison. All patients show SOX10+ cell depletion in the lesion core. C Co-immunohistochemistry for SOX10 (red) and MCT2 (green) in the white matter of a control subject. D Sections of of cerebellar tissue of the patient with MS harboring a chronic active lesion. Low power image shows Luxol fast blue (light blue) staining for myelin, Cresyl Violet (dark blue) staining for neuronal cell bodies, and MHC II (black) staining for inflammatory cells. Squares indicate areas analyzed on adjacent sections by immunofluorescence. Images of co-immunolabelling for SOX10 (red) and MOG (gray) show Sox10+ cells are present in the NAWM and perilesion but are largely depleted from the lesion core. Co-immunohistochemistry for SOX10 (red) and MCT2 (green) in the NAWM (E)and perilesion (F). G, H Quantification of MCT2/SOX10 colocalization. G The percentage of SOX10+ cells that express MCT2 in control subject white matter is significantly higher than in the MS NAWM, two-sided Welch’s unpaired t-test, Mean ± SEM. *p = 0.0246, and H Comparison of SOX10+ cells that express MCT2 between MS NAWM, perilesion and lesion core, Friedman test (P = 0.0093). Dunn’s multiple comparisons test detects significant differences between NAWM and the lesion core, *p = 0.0240. Controls N = 4 and MS patients N = 4. C, E, F Purple arrows: MCT2+cells; light blue arrows =MCT2- cells. Scale bars: C, E, F 20 µm, D 1mm, D insets 50 μm. NAWM normal appearing white matter. PL perilesion. L lesion core.

Fig 5: MCT2 deletion in myelinating oligodendrocytes using AAV-mediated Cre-Lox approach.A Schematic presentation of the experimental strategy employed to delete Slc16a7 in mature oligodendrocytes. Wildtype (wt) and MCT2lox/lox mice were injected with oligodendrotropic Olig001-AAV carrying either GFP or Cre-GFP construct in the spinal cord dorsal white matter. Mice were sacrificed at 3 and 6 weeks post injection. B Co-immunolabelling for GFP in green and mature oligodendrocyte marker APC/CC1 in red. Arrowheads indicate double-labelled cells. C Co-labeling for GFP (green) and Cre-recombinase (magenta) in MCT2lox/lox mice injected with Olig001-GFP or Olig001-Cre-GFP.Arrowheads indicate double-labelled cells, only apparent in Olig001-Cre-GFP injected MCT2lox/lox mice. D Co-immunolabelling for GFP in green and MCT2 in magenta. White arrows indicate GFP+ cells. Yellow arrows indicate GFP- cells. MCT2lox/lox mice injected with Olig001-GFP show comparable intensity of MCT2 labeling between GFP+ and GFP- cells. WT mice injected with Olig001-Cre-GFP also showcomparable intensity of MCT2 labeling between GFP+ and GFP- cells. In MCT2lox/lox mice injected with Olig001-Cre-GFP (AAV-MCT2KO mice), while GFP- cells show MCT2 staining comparable to the AAV-controls, only residual staining is observed on GFP+ cells. E The proportion of GFP+ cells that are MCT2+ significantly decreases in AAV-MCT2KO compared to AAV-controls. Two-sided Unpaired student t-test, Mean ± SEM (*) p = 0.0007. n = 4 mice per group. All scale bars=10 µm. Mouse drawing in A is from Pixabay https://pixabay.com/vectors/search/lab%20mouse/.