Fig 2: (A) Representative confocal images of nodes of Ranvier (Nav1.6; red) and paranodes (caspr; green) in corpus callosum of control and clemastine treated P21 mice. Scale bar = 5 µm. Rigth, histograms shows average node of Ranvier number and length in control and clemastine-treated mice (n = 6 per experimental group). (B) Representative confocal images of MBP+ myelin internodes (green) flanked by two caspr+ paranodes (red; arrows). Right, average and cumulative internode length distribution in cerebral cortex of control and clemastine-treated P21 mice (n = 6 per group). (C) Representative confocal images of MBP and Neurofilament (NF-L) immunostainings of the corpus callosum. Scale bar = 15 µm. Right, histograms show average immunoreactivity of MBP and NF-L staining in corpus callosum from control and clemastine-treated P21 mice (n = 6 per group).

Fig 3: Microglial processes are less engaged with axons following cFPI in the rat. Representative 3D reconstructions of Iba-1+ microglial (white) processes with (A) MBP+ intact myelinated axons (red) in sham-injured rats or APP+ injured axonal swellings (green) in cFPI rats either (B) 6 h or (C) 1 day post-injury. DAPI-labeled nuclei are in blue. Bar graphs depicting the average number of (D) total microglial process contacts, (E) terminal processes, (F) process cups, and (G) process passes of Iba-1+ microglial that contact either MBP+ intact fibers or APP+ injured axonal swellings following sham or TBI in the rat. Note that overall, total microglial process contacts are decreased in intact axonal segments at both 6 h and 1 day post-cFPI and in injured axonal swellings at 6 h post-injury in rats. Graphs depict the mean ± standard error of the mean. *p < 0.05 compared to sham; #p < 0.05 compared to injured axonal swellings 1 day post-cFPI; &p < 0.05 compared to intact axonal segments 6 h post-cFPI. Scale bar: 5 µm.

Fig 4: Structural changes of the Purkinje axon in the LES cerebellum (A). Calbindin (CB)-expressing Purkinje cells (green) in LE, LEHet, and LES cerebella. Caspr (blue) and NavPan (red) expression along Purkinje axons (arrowheads). (B) Purkinje axons expressing CB (green) in the white matter of the cerebellum of LE, LEHet, and LES. Arrowheads mark nodal and paranodal structures, expressing Caspr (blue) and NavPan (red), enlarged in inset. Inset width is 18 µm. While the nodal structure of Purkinje axons in LE and LEHet remain intact, Nav and Caspr expression in LES Purkinje axons is diffuse. (C) CB-expressing Purkinje axons (green) and myelin basic protein (MBP) expression (red) in the white matter of the cerebellum of LE, LEHet, and LES. MBP expression is absent in the LES cerebellum, where spheroid swellings were observed in Purkinje axons (arrowheads). (D) EM images of myelination around Purkinje axons in LE, LEHet, and LES. Myelin was thinner in LEHet than LE, and nearly absent in LES. (E) Diagram of a Purkinje cell with dendrites that extend into the molecular layer and an axon projecting through the granule cell layer and white matter that synapses in the DCN. Recording electrode (blue) represents the recording site in Fig. 2 through 4. Stimulating electrode (red) indicates the stimulating site in Fig. 4. (F) Number of Purkinje cells per 304.5 µm × 304.5 µm square was not different between LE, LEHet, and LES animals. (G) There were no significant differences between the length of NavPan staining along Purkinje AIS between LE, LEHet, and LES animals. (H) Quantification of the g-ratio is calculated by dividing the radius of the axon (blue) by the total radius of the axon and myelin (red). The g-ratio quantified from electron microscopy (EM) images was significantly higher in LEHet and LES compared to LE.

Fig 5: Plp1-targeting ASOs increase Mbp expression and rescue oligodendrocyte numbers in jimpy mice.a, Western blot data showing the level of MBP protein (n=3 mice). b, RT-qPCR data showing the level of Mbp transcript (n=3 mice). c, Western blot data showing the level of MBP (n=3 mice). d, Immunohistochemistry images with hematoxylin counterstain of whole brain sagittal sections showing MBP+ myelin. Scale bar, 1mm. e, Quantification of cleaved-caspase 3+ apoptotic cells (n=3 mice). f, Quantification of CC1+/Olig2+ oligodendrocytes (n=4 mice). g, Quantification of the number of Olig2+ glial lineage cells (n=4 mice). h, Quantification of the number of PDGFRa+/Olig2+ OPCs (n=4 mice).All data collected at 3 weeks post-ASO injection (30µg dose at birth). Individual data points represent the mean value of 4 technical replicates for each biological replicate (individual mice) (b) or independent biological replicates (individual mice) (a, c-h), indicated by open circles. Graph bars indicate mean ± standard deviation. p-values calculated using one-way ANOVA with Dunnett’s correction for multiple comparisons. p-values stated for p<0.1, otherwise not significant (n.s). See Supplementary Data 4 for full western blot images for all samples.