Fig 2: Quantification of myelinated axons after the delivery of neurotrophic factor-encoding vectors 8 weeks post-injury. mHA-MP scaffolds were delivered after injury containing BDNF, NT3, or control FLuc-encoding vectors (a) and showed NF200+ axons (b) and MBP+ oligodendrocytes (c) within the scaffolds. Zoomed-in images of the areas indicated by white boxes in (b) are shown in (d). White arrows indicate NF200+/MBP- axons, while pink arrows indicate NF200+/MBP+ axons. The delivery of vectors encoding for the BDNF increased the density of axons (NF200+) and myelinated axons (NF200+/MBP+). The axonal density was significantly higher with the delivery of the BDNF relative to NT3 (p < 0.1) but not FLuc control (e). The net orientation of axons was near parallel, relative to the longitudinal axis of the spinal cord, in each condition, with no significant differences among conditions (f). BDNF delivery led to increased proportion of myelinated axons (g) and densities of myelinated axons (h) relative to both NT3 and control ([p < 0.05, Kruskal-Wallis test with Dunn's multiple comparisons test, n = 5–6, scale bars = 200 µm for (a)–(c) and 20 µm for (d), white arrows = unmyelinated axon and pink arrows = myelinated axon].

Fig 3: Lineage plasticity of SVZ neuroblasts after LPC induced demyelination(A–C) LPC-induced demyelination of GAD67-GFP mouse, 6 days after injection. (A) GFP+ NB (green) ectopically migrate from the RMS toward the lesion site in the CC (dashed line). Higher magnification: GFP+ NB exiting the RMS. (B) Some GFP+ NB start expressing oligodendrocyte markers such as Olig2 (red). (C) Density of GAD67-GFP+ Olig2+ cells in the CC and SVZ, 3 and 6 dpi. Error bars: mean ± SEM *p < 0.05, **p < 0.01; Mann-Whitney test (n= 5 mice).(D and E) LPC-induced demyelination of DCX-CreERT2:YFP mouse. 7 days after LPC injection, YFP+ NB are identified in the lesion (dashed line) and some of them express the oligodendrocyte marker Olig2 (red).(F–H) LPC-induced demyelination of DCX-CreERT2:mTmG mouse. 3 weeks after LPC injection at the site of the lesion, GFP+ cells adopt a complex morphology and express the mature oligodendrocyte marker MBP (red in F) and GFP+ segments are flanked by Caspr staining indicating paranodal organization. (H) Electron micrograph showing GFP immunogold-labeled myelin sheaths. The presence of gold beads within compact myelin sheaths surrounding axons an attests for the differentiation of GFP+ NB into myelinating oligodendrocytes. Scale bars: 200 µm in A and D, 20 µm in B, 50 µm in E to G and 150 nm in H. SVZ: sub-ventricular zone, CC: corpus callosum, CX: cortex, LV: lateral ventricle.

Fig 4: Multi-focal lesions in the brain of aged TWI treated with AAV9-GALC(A–D) MBP-stained sagittal whole-brain scans of P40 WT (A), sham P40 TWI (B), aged TWI+AAV (C), and aged TWI+AAV+BMT (D). In both TWI+AAV (C) and TWI+AAV+BMT (D), multi-focal demyelinating lesions were observed. Tissue adjacent to the lateral ventricle (arrows) was affected in all aged animals examined; brainstem (arrowheads), corpus callosum, and cerebellum were also commonly affected. (E–H) Higher-magnification images of MBP-stained P40 WT (E), sham P40 TWI (F), P40 TWI+AAV (G), and P40 TWI+AAV+BMT (H). At this age, neither TWI+AAV nor TWI+AAV+BMT animals display any demyelinating lesions. (I–K) Higher-magnification images of MBP-stained aged WT (I), aged TWI+AAV (J), and aged TWI+AAV+BMT (K). The demyelinating lesions are evident in the stria terminals near the lateral ventricle (arrows) and corpus callosum (arrowheads). (L–O) Electron microscopy imaging was used to examine myelin structure between aged WT (L), sham P40 TWI (M), P40 TWI+AAV (N), and an aged TWI+AAV (O). No evidence of demyelination was detected in P40 TWI+AAV at P40 (N), displaying similar structure to a WT animal (L). Areas of demyelination in aged TWI+AAV (O) were similar in appearance to the diffuse demyelination seen in sham TWI (M); however, the damage was localized and displayed sharp demarcations between affected and unaffected tissue (O, dashed line, arrows). Evidence of axonal stress was also detected (arrowheads). Scale bars, (A)–(D) 1.5 mm; (E)–(K) 100 µm; (L)–(O) 2 µm.

Fig 5: The pH dependence of the relative proteolytic activity of IgGmix in the hydrolysis of MBP. The relative proteolytic activity corresponding to complete hydrolysis of MBP after 20 h of incubation with 0.2 mg/mL IgGmix was taken as 100%.