mTOR Signaling Regulates Metabolic Function in Oligodendrocyte Precursor Cells and Promotes Efficient Brain Remyelination in the Cuprizone Model

Abstract

In demyelinating diseases, such as multiple sclerosis, primary loss of myelin and subsequent neuronal degeneration throughout the CNS impair patient functionality. While the importance of mechanistic target of rapamycin (mTOR) signaling during developmental myelination is known, no studies have yet directly examined the function of mTOR signaling specifically in the oligodendrocyte (OL) lineage during remyelination. Here, we conditionally deleted Mtor from adult oligodendrocyte precursor cells (OPCs) using Ng2-Cre^ERT in male adult mice to test its function in new OLs responsible for remyelination. During early remyelination after cuprizone-induced demyelination, mice lacking mTOR in adult OPCs had unchanged OL numbers but thinner myelin. Myelin thickness recovered by late-stage repair, suggesting a delay in myelin production when Mtor is deleted from adult OPCs. Surprisingly, loss of mTOR in OPCs had no effect on efficiency of remyelination after lysophosphatidylcholine lesions in either the spinal cord or corpus callosum, suggesting that mTOR signaling functions specifically in a pathway dysregulated by cuprizone to promote remyelination efficiency. We further determined that cuprizone and inhibition of mTOR cooperatively compromise metabolic function in primary rat OLs undergoing differentiation. Together, our results support the conclusion that mTOR signaling in OPCs is required to overcome the metabolic dysfunction in the cuprizone-demyelinated adult brain.SIGNIFICANCE STATEMENT Impaired remyelination by oligodendrocytes contributes to the progressive pathology in multiple sclerosis, so it is critical to identify mechanisms of improving remyelination. The goal of this study was to examine mechanistic target of rapamycin (mTOR) signaling in remyelination. Here, we provide evidence that mTOR signaling promotes efficient remyelination of the brain after cuprizone-mediated demyelination but has no effect on remyelination after lysophosphatidylcholine demyelination in the spinal cord or brain. We also present novel data revealing that mTOR inhibition and cuprizone treatment additively affect the metabolic profile of differentiating oligodendrocytes, supporting a mechanism for the observed remyelination delay. These data suggest that altered metabolic function may underlie failure of remyelination in multiple sclerosis lesions and that mTOR signaling may be of therapeutic potential for promoting remyelination.

Keywords: cuprizone; glycolysis; mTOR; mitochondria; oligodendrocyte; remyelination.

Figure 1.

Mtor deletion from adult OPCs does not impair differentiation during remyelination after CPZ demyelination. A, Schematic of experimental timeline. B, Representative images of CC1 (red) and SOX10 (blue) immunostaining in control and Ng2-Mtor cKO callosa at 6 wpi + 2 wpr CTL diet, 6 wpi CPZ diet, and 6 wpi + 2 wpr CPZ diet. Scale bar, 50 µm. C, Quantification of SOX10⁺ OL lineage cells in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 2 wpr CTL diet, 6 wpi CPZ diet, and 6 wpi + 2 wpr CPZ diet; n = 3 or 4/group. D, Quantification of CC1^– SOX10⁺ OPCs in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 2 wpr CTL diet, 6 wpi CPZ diet, and 6 wpi + 2 wpr CPZ diet; n = 3 or 4/group. E, Quantification of CC1⁺ SOX10⁺ mature OLs in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 2 wpr CTL diet, 6 wpi CPZ diet, and 6 wpi + 2 wpr CPZ diet; n = 3 or 4/group. F, % of all OL lineage cells that are CC1⁺ mature OLs in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 2 wpr CTL diet, 6 wpi CPZ diet, and 6 wpi + 2 wpr CPZ diet; n = 3 or 4/group. *p ≤ 0.05.

Figure 2.

Area of remyelination is unaffected by Mtor deletion from adult OPCs after CPZ demyelination. A, Representative images of MOG immunostaining in control and Ng2-Mtor cKO callosa at 6 wpi + 2 wpr CTL diet, 6 wpi CPZ diet, and 6 wpi + 2 wpr CPZ diet. Scale bar, 500 µm. B, Quantification of the % of the callosum positive for MOG in control (black) and Ng2-Mtor cKO (red) at 6 wpi + 2 wpr CTL diet, 6 wpi CPZ diet, and 6 wpi + 2 wpr CPZ diet; n = 4-7/group.

Figure 3.

Ng2-Mtor cKO mice display delayed myelin wrapping during remyelination compared with controls after CPZ demyelination. A, Representative electron micrographs of control and Ng2-Mtor cKO callosa at 6 wpi CPZ diet. B, Quantification of average g-ratios in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi CPZ diet; n = 3 or 4/group. C, Scatter plot of control (black) and Ng2-Mtor cKO (red) g-ratios at 6 wpi CPZ diet; n > 700 axons/group. D, Representative electron micrographs of control and Ng2-Mtor cKO callosa at 6 wpi + 2 wpr CPZ diet. E, Quantification of average g-ratios in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 2 wpr CPZ diet; n = 3/group. F, Scatter plot of control (black) and Ng2-Mtor cKO (red) g-ratios at 6 wpi + 2 wpr CPZ diet; n > 800 axons/group. G, Representative electron micrographs of control and Ng2-Mtor cKO callosa at 6 wpi + 4 wpr CPZ diet. Scale bars: A, D, G, 1 µm. A, D, G, Red arrows point to representative similarly sized axons. H, Quantification of average g-ratios in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 4 wpr CPZ diet; n = 3/group. I, Scatter plot of control (black) and Ng2-Mtor cKO (red) g-ratios at 6 wpi + 4 wpr CPZ diet; n > 600 axons/group. J, g-ratio relative frequency diagram of control (black) and Ng2-Mtor cKO (red) g-ratios at 6 wpi CPZ diet. K, g-ratio relative frequency diagram of control (black) and Ng2-Mtor cKO (red) g-ratios at 6 wpi + 2 wpr CPZ diet. L, g-ratio relative frequency diagram of control (black) and Ng2-Mtor cKO (red) g-ratios at 6 wpi + 4 wpr CPZ diet. M, Quantification of the number of myelinated axons in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi CPZ diet; n = 3 or 4/group. N, Quantification of the number of myelinated axons in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 2 wpr CPZ diet; n = 3/group. O, Quantification of the number of myelinated axons in control (black) and Ng2-Mtor cKO (red) callosa at 6 wpi + 4 wpr CPZ diet; n = 3/group. *p ≤ 0.05. **p ≤ 0.01. ****p ≤ 0.0001.

Figure 4.

Mtor deletion from adult OPCs does not impair differentiation during remyelination after LPC demyelination of the spinal cord. A, Schematic of experimental timeline. B, Representative images of CC1 (red) and SOX10 (blue) immunostaining in control and Ng2-Mtor cKO 14 dpl dorsal and ventral LPC lesions. Scale bar, 50 µm. C, Quantification of SOX10⁺ OL lineage cells in control (black) and Ng2-Mtor cKO (red) 14 dpl dorsal and ventral lesions; n = 3 or 4/group. D, Quantification of CC1^– SOX10⁺ OPCs in control (black) and Ng2-Mtor cKO (red) 14 dpl dorsal and ventral lesions; n = 3 or 4/group. E, Quantification of CC1⁺ SOX10⁺ mature OLs in control (black) and Ng2-Mtor cKO (red) 14 dpl dorsal and ventral lesions; n = 3 or 4/group. F, % of all OL lineage cells that are CC1⁺ mature OLs in control (black) and Ng2-Mtor cKO (red) 14 dpl dorsal and ventral lesions; n = 3 or 4/group.

Figure 5.

Area of remyelination in spinal cord LPC lesions is unaffected by Mtor deletion from adult OPCs. A, Representative images of MBP (yellow represents dorsal lesions; red represents ventral lesions) and DAPI (blue) immunostaining in control and Ng2-Mtor cKO 14 dpl dorsal and ventral LPC lesions. Scale bar, 100 µm. White lines indicate lesions. B, Quantification of the % of the lesion positive for MBP in control (black) and Ng2-Mtor cKO (red) 14 dpl dorsal lesions; n = 4 or 5/group. C, Quantification of the % of the lesion positive for MBP in control (black) and Ng2-Mtor cKO (red) 14 dpl ventral lesions; n = 3/group. D, Representative images of MOG immunostaining in control and Ng2-Mtor cKO 14 dpl dorsal and ventral LPC lesions. Scale bar, 100 µm. E, Quantification of the % of the lesion positive for MOG in control (black) and Ng2-Mtor cKO (red) 14 dpl dorsal lesions; n = 4/group. F, Quantification of the % of the lesion positive for MOG in control (black) and Ng2-Mtor cKO (red) 14 dpl ventral lesions; n = 3/group.

Figure 6.

Remyelination thickness in Ng2-Mtor cKO spinal cord LPC lesions is not affected comparably to CPZ demyelination. A, Representative electron micrographs of control and Ng2-Mtor cKO 21 dpl dorsal LPC lesions. Scale bar, 4 µm. B, Quantification of average g-ratios in control (black) and Ng2-Mtor cKO (red) 21 dpl dorsal lesions; n = 3/group. C, Representative electron micrographs of control and Ng2-Mtor cKO 21 dpl ventral LPC lesions. Scale bar, 2 µm. D, Quantification of average g-ratios in control (black) and Ng2-Mtor cKO (red) 21 dpl ventral lesions; n = 3/group. E, Scatter plot of control (black) and Ng2-Mtor cKO (red) 21 dpl dorsal lesion g-ratios; n > 300 axons/group. F, g-ratio relative frequency diagram of control (black) and Ng2-Mtor cKO (red) 21 dpl dorsal lesion g-ratios. G, Scatter plot of control (black) and Ng2-Mtor cKO (red) 21 dpl ventral lesion g-ratios; n > 400 axons/group. H, g-ratio relative frequency diagram of control (black) and Ng2-Mtor cKO (red) 21 dpl ventral lesion g-ratios. I, Quantification of the number of myelinated axons in control (black) and Ng2-Mtor cKO (red) dorsal white matter at 21 dpl; n = 3/group. J, Quantification of the number of myelinated axons in control (black) and Ng2-Mtor cKO (red) ventral white matter at 21 dpl; n = 3/group. *p ≤ 0.05.

Figure 7.

GFP⁺ OLs contribute to remyelination in demyelinated brain and spinal cord. A, qRT-PCR of Mtor gene transcription normalized to β-actin in isolated NG2⁺ PDGFRα⁺ OPCs from control (black) and Ng2-Mtor cKO (red) adult brains; n = 3 pools of 3 samples/group. B, Corresponding graphical difference between the means from Student's t test for brain OPCs. C, qRT-PCR of Mtor gene transcription normalized to β-actin in isolated NG2⁺ PDGFRα⁺ OPCs from control (black) and Ng2-Mtor cKO (red) adult spinal cords; n = 3 pools of 3 samples/group. D, Corresponding graphical difference between the means from Student's t test for spinal cord OPCs. E, Representative images of MBP (blue) and GFP (green) immunostaining in control and Ng2-Mtor cKO brains at 6 wpi CPZ. Scale bar, 100 µm. F, Representative images of MBP (blue) and GFP (green) immunostaining in control and Ng2-Mtor cKO spinal cords at 14 dpl LPC. Scale bar, 100 µm. *p ≤ 0.05.

Figure 8.

Ng2-Mtor cKO remyelination is not delayed after LPC demyelination of the callosum. A, Schematic of experimental timeline. B, Representative electron micrographs of control and Ng2-Mtor cKO 14 dpl callosum LPC lesions. Scale bar, 1 µm. Red arrows indicate axons of similar diameter. C, Quantification of average g-ratios in control (black) and Ng2-Mtor cKO (red) 14 dpl callosum lesions; n = 3 or 4/group. D, Scatter plot of control (black) and Ng2-Mtor cKO (red) 14 dpl callosum lesion g-ratios; n > 400 axons/group. E, g-ratio relative frequency diagram of control (black) and Ng2-Mtor cKO (red) 14 dpl callosum lesion g-ratios. F, Quantification of the number of myelinated axons in control (black) and Ng2-Mtor cKO (red) corpus callosum at 14 dpl; n = 3 or 4/group.

Figure 9.

Delayed remyelination in Ng2-Mtor cKO brains requires the presence of CPZ during early repair. A, Schematic of experimental timeline. B, Representative electron micrographs of control and Ng2-Mtor cKO callosa at 4 wpi + 2 wpr CPZ diet. Scale bar, 1 µm. Red arrows point to axons of similar diameter. C, Quantification of average g-ratios in control (black) and Ng2-Mtor cKO (red) callosa at 4 wpi + 2 wpr CPZ diet; n = 3 or 4/group. D, Scatter plot of control (black) and Ng2-Mtor cKO (red) g-ratios at 4 wpi + 2 wpr CPZ diet; n > 600 axons/group. E, g-ratio relative frequency diagram of control (black) and Ng2-Mtor cKO (red) g-ratios at 4 wpi + 2 wpr CPZ diet. *p ≤ 0.0001.

Figure 10.

OLs treated in vitro with either CPZ or RAPA exhibit impaired mitochondrial function. A, A representative trace of OCR in differentiating primary rat OPCs treated with either vehicle (black) or 50 μm CPZ (blue) for 3 d. Dashed lines indicate the time points of sequential addition of compounds oligomycin, FCCP, and rotenone/antimycin A. B, Corresponding basal respiration graph for A. C, Corresponding maximal respiration graph for A. D, A representative trace of ECAR in differentiating primary rat OPCs treated with either vehicle (black) or 50 μm CPZ (blue) for 3 d. E, Corresponding basal glycolysis graph for C. F, Corresponding glycolytic capacity graph for C. G, A representative trace of OCR in differentiating primary rat OPCs treated with either vehicle (black) or 10 nm RAPA (orange) for 3 d. Dashed lines indicate the time points of sequential addition of compounds oligomycin, FCCP, and rotenone/antimycin A. H, Corresponding basal respiration graph for G. I, Corresponding maximal respiration graph for G. J, A representative trace of ECAR in differentiating primary rat OPCs treated with either vehicle (black) or 10 nm RAPA (orange) for 3 d. K, Corresponding basal glycolysis graph for J. L, Corresponding glycolytic capacity graph for J. Data are mean ± SD. ***p ≤ 0.001. ****p ≤ 0.0001.

Figure 11.

CPZ and RAPA additively affect mitochondrial function in differentiating OLs in vitro. A, A representative trace of OCR in differentiating primary rat OPCs treated with either 10 nm RAPA (orange) or 50 μm CPZ + 10 nm RAPA (green) for 3 d. Dashed lines indicate the time points of sequential addition of compounds oligomycin, FCCP, and rotenone/antimycin A. B, Corresponding basal respiration graph for A. C, Corresponding maximal respiration graph for A. D, A representative trace of ECAR in differentiating primary rat OPCs treated with either 10 nm RAPA (orange) or 50 μm CPZ + 10 nm RAPA (green) for 3 d. E, Corresponding basal glycolysis graph for C. F, Corresponding glycolytic capacity graph for C. G, A representative trace of OCR in differentiating primary rat OPCs treated with either 50 μm CPZ (blue) or 50 μm CPZ + 10 nm RAPA (green) for 3 d. Dashed lines indicate the time points of sequential addition of compounds oligomycin, FCCP, and rotenone/antimycin A. H, Corresponding basal respiration graph for G. I, Corresponding maximal respiration graph for G. J, A representative trace of ECAR in differentiating primary rat OPCs treated with either 50 μm CPZ (blue) or 50 μm CPZ + 10 nm RAPA (green) for 3 d. K, Corresponding basal glycolysis graph for J. L, Corresponding glycolytic capacity graph for J. Data are mean ± SD. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001.