Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination

Abstract

Although the mammalian target of rapamycin (mTOR) is an essential regulator of developmental oligodendrocyte differentiation and myelination, oligodendrocyte-specific deletion of tuberous sclerosis complex (TSC), a major upstream inhibitor of mTOR, surprisingly also leads to hypomyelination during CNS development. However, the function of TSC has not been studied in the context of remyelination. Here, we used the inducible Cre-lox system to study the function of TSC in the remyelination of a focal, lysolecithin-demyelinated lesion in adult male mice. Using two different mouse models in which Tsc1 is deleted by Cre expression in oligodendrocyte progenitor cells (OPCs) or in premyelinating oligodendrocytes, we reveal that deletion of Tsc1 affects oligodendroglia differently depending on the stage of the oligodendrocyte lineage. Tsc1 deletion from NG2⁺ OPCs accelerated remyelination. Conversely, Tsc1 deletion from proteolipid protein (PLP)-positive oligodendrocytes slowed remyelination. Contrary to developmental myelination, there were no changes in OPC or oligodendrocyte numbers in either model. Our findings reveal a complex role for TSC in oligodendrocytes during remyelination in which the timing of Tsc1 deletion is a critical determinant of its effect on remyelination. Moreover, our findings suggest that TSC has different functions in developmental myelination and remyelination.SIGNIFICANCE STATEMENT Myelin loss in demyelinating disorders such as multiple sclerosis results in disability due to loss of axon conductance and axon damage. Encouragingly, the nervous system is capable of spontaneous remyelination, but this regenerative process often fails. Many chronically demyelinated lesions have oligodendrocyte progenitor cells (OPCs) within their borders. It is thus of great interest to elucidate mechanisms by which we might enhance endogenous remyelination. Here, we provide evidence that deletion of Tsc1 from OPCs, but not differentiating oligodendrocytes, is beneficial to remyelination. This finding contrasts with the loss of oligodendroglia and hypomyelination seen with Tsc1 or Tsc2 deletion in the oligodendrocyte lineage during CNS development and points to important differences in the regulation of developmental myelination and remyelination.

Keywords: NG2; TSC; adult OPCs; mTOR; remyelination.

Figure 1.

Tsc1 deletion from NG2⁺ cells increases mTORC1 signaling, but does not affect oligodendrocyte differentiation. A, Timeline of tamoxifen administration, lysolecithin injection, and sample collection. B, Expression of GFP reporter in NG2-Tsc1cKO lesion at 14 dpl. Scale bar, 100 μm. C, Colabeling of Sox10, CC1, and GFP in 14 dpl lesion of NG2-Tsc1cKO animal. Scale bar, 10 μm. D, Representative images from control and NG2-Tsc1cKO animals of pS6RP expression in Sox10⁺ cells at 14 dpl. Scale bar, 100 μm. E, Representative Western blot of TSC1 from control and NG2-Tsc1cKO animals. F, Colabeling of Sox10 and CC1 at 14 dpl in the lesions of control and NG2-Tsc1cKO animals. Scale bar, 100 μm. G, H, Quantification of number of Sox10⁺ cells/mm² (G) and percentage of Sox10⁺ cells that were also CC1⁺ (H) within the lesion at 7 and 14 dpl. For 7 dpl, n = 5 control, n = 3 NG2-Tsc1cKO; for 14 dpl, n = 4/group. All values are expressed as ± SEM.

Figure 2.

Initial lesion size and microgial/macrophage response are unchanged with Tsc1 deletion from NG2⁺ cells. A, Representative image of Iba1 staining and GFP reporter in the lesion of an NG2-Tsc1cKO animal. Arrows indicate GFP⁺ cells, all of which are negative for Iba1. Scale bar, 50 μm. B, Left two panels depict colocalization of pericyte marker PDGFRβ with GFP reporter in NG2-Tsc1cKO lesion. Solid arrowheads identify colocalization; hollow arrowheads identify PDGFRβ⁺/GFP-cells. Rightmost panel shows low expression of TSC1 in PDGFRβ-positive pericytes in control animals. Scale bars, 50 μm. C, Representative images from control and NG2-Tsc1cKO animals of Luxol fast blue staining of the dorsal column at 7 dpl. Dotted line denotes borders of dorsal white matter. Scale bar, 100 μm. D, Immunostaining for Iba1 at 7 dpl in control and NG2-Tsc1cKO animals. Scale bar, 100 μm.

Figure 3.

Increased myelin protein expression in NG2-Tsc1cKO animals during remyelination. A, Immunostaining for myelin proteins MBP and MOG in the lesion of control and NG2-Tsc1cKO animals. Representative images for MBP and MOG are from 14 and 21 dpl, respectively. Scale bars, 100 μm. B, Analysis of the percentage of the lesioned area that was MBP⁺ or MOG⁺ at 14 and 21 dpl. Two-way ANOVA analyses revealed a significant main effect of genotype on MOG expression (*p < 0.05) and MBP expression (**p < 0.01) with a significant difference in MBP expression at 14 dpl between control and NG2-Tsc1cKO animals (**p < 0.01). n = 3 − 5 animals/group/time point. All values are expressed as ± SEM.

Figure 4.

Remyelination is accelerated in NG2-Tsc1cKO animals. A, Low (top) and high (bottom) magnification images of toluidine blue-stained semithin sections of control and NG2-Tsc1cKO animals at 21 dpl. Low-magnification images capture the entire lesioned dorsal white matter; high-magnification images show remyelinating axons at the lesion border. Scale bars: top, 100 μm; bottom, 20 μm. B, Rank analysis of toluidine blue images by three independent, blinded rankers with higher ranks indicating greater remyelination. Each symbol represents an independent section; two to three independent sections/animal were used in the ranking. Lines indicate median rank. n = 6 control animals, n = 5 NG2-Tsc1 cKO animals. C, Representative EMs from lesioned spinal cord of control and NG2-Tsc1cKO animals at 21 dpl. Arrows and arrowheads denote axons of similar axon diameter representing axons of large (≥1.5 μm) and small (<1.5 μm) diameter, respectively. Scale bar, 4 μm. D, Quantification of myelin thickness through g-ratio analysis at 21 dpl. The g-ratios of individual axons were plotted in relation to their diameter. Control animals are represented by the solid trend line; NG2-Tsc1cKO animals by the dashed trend line. ****p < 0.0001, linear regression analysis. E, G-ratios were compared within small-diameter (<1.5 μm) and large-diameter (≥1.5 μm) axons. *p < 0.05, Holm–Sidak test. All values are expressed as ± SEM. F, G-ratios were quantified in control and NG2-Tsc1cKO animals at 49 dpl. Linear regression analysis revealed no significant differences between the two trend lines (control, solid trend line; NG2-Tsc1cKO, dashed trend line). At both time points, g-ratio measurements were performed on >100 axons from two different regions of the remyelinated lesion in each animal. n = 3/group.

Figure 5.

Tsc1 deletion from differentiated oligodendrocytes has no effect on oligodendrocyte number or myelin protein expression. A, Timeline of lysolecithin injection, tamoxifen injections, and sample collection. B, Representative images from control and PLP-Tsc1cKO animals of TSC1 expression in Sox10⁺ cells. Solid arrowheads identify colocalization; hollow arrowheads identify Sox10⁺ cells that are negative for TSC1. Scale bar, 50 μm. C, Quantification of the number of Sox10⁺ cells that have high TSC1 expression in PLP-Tsc1cKO animals compared with controls (**p < 0.01). n = 4/group. D, Representative Western blots of TSC1 expression in O4⁺ cells isolated from control and PLP-Tsc1cKO animals. E, Colabeling of Olig2 and CC1 in lesions from control and PLP-Tsc1cKO animals. Scale bar, 50 μm. F, Quantification of the total number of Olig2⁺ cells and CC1⁺/Olig2⁺ cells. n = 4/group. G, Immunostaining for myelin proteins MBP (top) and MOG (bottom). Solid lines denote lesion boundaries; dotted lines denote boundaries of dorsal white matter. Scale bars, 100 μm. H, Quantification of the percentage of lesioned area that was MBP⁺ or MOG⁺ at 21 dpl in control and PLP-Tsc1cKO animals. n = 5 control, 4 PLP-Tsc1cKO. NS, Not significant. All values are expressed as ± SEM.

Figure 6.

Remyelination is slower in PLP-Tsc1 cKO animals. A, Representative images of toluidine blue-stained semithin sections from 21 dpl control and PLP-Tsc1cKO animals at low (top) and high (bottom) magnification. Low-magnification images capture the entire lesioned dorsal white matter; high-magnification images show remyelinating axons at the lesion border. Scale bars: top, 100 μm; bottom, 20 μm. B, Rank analysis of remyelination in toluidine blue images with the least remyelinated section receiving a rank of 1. Each symbol represents an individual section; two independent sections were analyzed per animal. Each color represents a different ranker. Lines indicate median rank. n = 6 control animals, n = 4 PLP-Tsc1cKO animals. C, D, Representative EMs from control and PLP-Tsc1cKO animals and g-ratio scatterplots at 21 dpl (C) and 49 dpl (D). Scale bar, 4 μm. Control animals are represented by the solid trend line; PLP-Tsc1cKO animals by the dashed trend line. Linear regression analysis revealed a significantly higher y-intercept for the PLP-Tsc1cKO trend line at 21 dpl, ****p < 0.0001. No differences were seen between control and PLP-Tsc1cKO animals in g-ratios across axon diameters at 49 dpl.

Figure 7.

Stage-dependent effects of Tsc1 deletion on remyelination. Comparison of remyelination in control (middle row) animals and NG2-Tsc1 cKO (top row) or PLP-Tsc1 cKO (bottom row) animals reveal differential effects of Tsc1 deletion in the two different paradigms. Before lysolecithin (left column), the NG2-Tsc1 cKO receive tamoxifen, so NG2⁺ OPCs are recombined and express the Cre reporter GFP. At 21 dpl (right column), a time point associated with robust remyelination, the nonremyelinated lesioned area is smaller and the larger-diameter remyelinated axons within the lesion have thicker myelin in the NG2-Tsc1 cKO animals compared with control. PLP-Tsc1 cKO animals received tamoxifen 8–13 dpl; therefore, the remyelinating oligodendrocytes are now GFP⁺ recombined cells from which the Tsc1 allele has been excised. At 21 dpl, the nonremyelinated area of the lesion is larger and the remyelinated axons have thinner myelin in the PLP-Tsc1 cKO animals compared with controls. Green-colored cells represent cells that have undergone recombination and the GFP reporter is consequently expressed.