ERK1/2 Activation in Preexisting Oligodendrocytes of Adult Mice Drives New Myelin Synthesis and Enhanced CNS Function

Increased pERK1/2 in adult OPCs, but not preexisting OLs, does not cause global hypermyelination. A, Immunostaining of Pdgfrα-Cre^ERT;Mek1DD/+ corpus callosum with antibodies against NG2 (red) and GFP (green) confirms that adult OPCs have recombined to activate the expression of MEK1DD and the GFP reporter. DAPI marks cell nuclei in blue. Scale bar, 10 μm. B, CC1, GFP, and pERK triple immunostaining of 2 mpi Pdgfrα-Cre^ERT;Mek1DD/+ corpus callosum confirms that MEK1DD expressing adult OPCs differentiate into mature oligodendrocytes with elevated pERK1/2. DAPI marks cell nuclei in blue. Scale bar, 10 μm. C, MBP immunostaining of matched spinal cord sections from WT;Mek1DD/+ and Pdgfra-Cre^ERT;Mek1DD/+ mice at 2 mpi demonstrates that there is no significant expansion of the white matter tracts. Scale bar, 500 μm. D, Electron micrographs from the corpus callosum at 2 mpi reveals comparable myelin thickness in WT;Mek1DD/+ and Pdgfra-Cre^ERT;Mek1DD/+ mice. Scale bar, 2 μm. Arrows indicate representative axons. E, Scatter plot depicting similar g-ratios from WT;Mek1DD/+ (blue) and Pdgfra-Cre^ERT;Mek1DD/+ (red) mice in relation to axon diameter. F, Immunostaining with antibodies against MBP and mGFP reveals thicker myelin produced by a recombined newly generated OL versus an unrecombined OL. Scale bar, 1 μm. At least three mice per genotype were used for all analyses; at least 100 axons per mouse were analyzed for g-ratio calculations.

Enhanced ERK1/2 signaling in mature OLs of adult mice drives the synthesis of excess myelin proteins and lipids. A, B, Western blot analysis of corpus callosum and overlying cortex at 2 mpi reveals increased levels of the myelin proteins MOG, PLP, and the 18.5 kDa isoform of MBP in Plp-Cre^ERT;Mek1DD/+ (MUT) mice compared with WT;Mek1DD/+ (WT) littermate controls. C, qRT-PCR from corpus callosum and overlying cortex at 2 mpi shows significantly increased Mog and Plp transcripts in MUT (red) mice compared with WT (blue) controls. D, Quantification of the major myelin lipid GalC species using mass spectrometry shows a significant increase in the relative concentration of GalC in MUT (red) compared with WT (blue) corpus callosum, each d18:1 species is normalized to itself in WT. E, qRT-PCR results demonstrate a significant upregulation of Myrf and Cgt at 2 mpi in MUT (red) mice. F, Similar 1RT-PCR analysis at an earlier time point of 14 dpi indicates a significant increase in Cgt, but not Myrf, in MUT (red) corpus callosum compared with littermate (blue) controls. ***p < 0.001, **p < 0.01, *p < 0.05. Data are shown as mean ± SEM. Four to five mice per genotype were used for Western blot analysis; at least three mice per genotype were used for qRT-PCR and LC-MS/MS analysis.

ERK1/2-mediated hypermyelination results in reciprocal changes to the length of the nodal and paranodal regions. A, CASPR (red) and NaV1.6 (green) immunostaining of matched sections of corpus callosum from WT;Mek1DD/+ (WT) and Plp-Cre^ERT;Mek1DD/+ (MUT) littermates at 2 mpi. Scale bar, 5 μm. B, Quantification of the number of nodes of Ranvier demonstrates a comparable overall density of nodes in WT and MUT mice. C, Measurement of the length of CASPR staining indicates an increase in paranode length in MUT mice, whereas measurement of NaV1.6 staining (D) reveals a concurrent decrease in node length in MUT corpus callosum. E, High-magnification electron micrographs of representative paranodes from corpus callosum demonstrate a comparable ultrastructure of WT and MUT paranodes. Ax, Axon; N, node; P, paranode. Scale bars, 400 nm. *p < 0.05. Data are shown as mean ± SEM. At least three mice per genotype were used for all analyses; at least 100 nodes or paranodes per mouse were measured for length analysis.

Plp-Cre^ERT;Mek1DD/+ mice exhibit increased CV in the CNS that does not significantly affect motor function but does enhance specific forms of hippocampal-based learning. A, Average ABR traces from WT;Mek1DD/+ (WT, blue) and Plp-Cre^ERT;Mek1DD/+ (MUT, red) mice at 2 mpi after 80 dB click stimulation show a visible leftward shift of the peaks in MUT mice, demonstrating increased conduction speed along axons. B, Quantification of results shown in A reveals a statistically significant decrease in the absolute latency to peaks II, III, and V of MUT (red) mice compared with WT (blue) controls. C, Interpeak latency after 16 kHz stimulation between peaks II–IV is also decreased in MUT (red) mice. D, Data from open-field test showing no differences in the distance (cm) traveled over blocks of time (5 min each) between WT (blue) and MUT (red) mice at 2 mpi. E, Complex ladder analysis shows no significant differences in the number of foot slips made by WT (blue) MUT (red) mice over three consecutive trials. F, Rotarod testing shows no differences between WT (blue) and MUT (red) mice in their latency to fall over the course of 5 training days, revealing no differences in motor coordination or learning. G, NOR testing shows no difference in percentage time exploring the novel object between WT (blue) and MUT (red) mice, suggesting no differences in novelty discrimination. H, Conditioned fear data showing no difference in percentage freezing between WT (blue) and MUT (red) mice in the cue trial, but indicating a significant difference in percentage freezing between WT and MUT mice in the context trial. **p < 0.01, *p < 0.05. Data are shown as mean ± SEM. Ten mice of each genotype were used for most analyses, including ABR trials; at least seven mice of each genotype were used for behavioral analyses.

Sustained activation of ERK1/2 in mature oligodendrocytes does not alter the immediate consequences of toxin-induced demyelination. A, Immunostaining with antibodies against mGFP at 2 dpl indicates a loss of preexisting myelin (demyelination) leading to a lesioned area within the dorsal column of the spinal cord (SC). Scale bar, 100 μm. Dotted lines demarcate the dorsal column. B, MBP immunostaining at 2 dpl confirms an equivalent demyelinated area in the dorsal column of the SC in Plp-Cre^ERT;+/+;mGFP and Plp-Cre^ERT;Mek1DD/+;mGFP mice. Scale bar, 100 μm. Dotted elliptical demarcates the lesioned area. C, Colabeling the lesioned dorsal column with EdU and OLIG2 reveals EdU+, OLIG2+-colabeled cells. Scale bar, 50 μm. D, E, Sustained activation of ERK1/2 in OLs does not lead to a change in the total number of OLIG2+ cells or in the percentage of EdU+, OLIG2+-colabeled cells divided by the total number of OLIG2+ cells at 2 dpl, revealing no differences in OL survival or OPC proliferation between Plp-Cre^ERT;+/+;mGFP (blue) and Plp-Cre^ERT;Mek1DD/+;mGFP (red) mice. Three to five mice per genotype were used for all analyses.