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. 2013 Jul 17;33(29):11899-915.
doi: 10.1523/JNEUROSCI.1131-13.2013.

Effects of Adult Neural Precursor-Derived Myelination on Axonal Function in the Perinatal Congenitally Dysmyelinated Brain: Optimizing Time of Intervention, Developing Accurate Prediction Models, and Enhancing Performance

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Free PMC article

Effects of Adult Neural Precursor-Derived Myelination on Axonal Function in the Perinatal Congenitally Dysmyelinated Brain: Optimizing Time of Intervention, Developing Accurate Prediction Models, and Enhancing Performance

Crystal A Ruff et al. J Neurosci. .
Free PMC article

Abstract

Stem cell repair shows substantial translational potential for neurological injury, but the mechanisms of action remain unclear. This study aimed to investigate whether transplanted stem cells could induce comprehensive functional remyelination. Subventricular zone (SVZ)-derived adult neural precursor cells (aNPCs) were injected bilaterally into major cerebral white matter tracts of myelin-deficient shiverer mice on postnatal day (P) 0, P7, and P21. Tripotential NPCs, when transplanted in vivo, integrated anatomically and functionally into local white matter and preferentially became Olig2+, Myelin Associated Glycoprotein-positive, Myelin Basic Protein-positive oligodendrocytes, rather than Glial Fibrillary Acidic Protein-positive astrocytes or Neurofiliment 200-positive neurons. Processes interacted with axons and transmission electron microscopy showed multilamellar axonal ensheathment. Nodal architecture was restored and by quantifying these anatomical parameters a computer model was generated that accurately predicted action potential velocity, determined by ex vivo slice recordings. Although there was no obvious phenotypic improvement in transplanted shi/shis, myelinated axons exhibited faster conduction, lower activation threshold, less refractoriness, and improved response to high-frequency stimulation than dysmyelinated counterparts. Furthermore, they showed improved resilience to ischemic insult, a promising finding in the context of perinatal brain injury. This study describes, for the first time mechanistically, the functional characteristics and anatomical integration of nonimmortalized donor SVZ-derived murine aNPCs in the dysmyelinated brain at key developmental time points.

Figures

Figure 1.
Figure 1.
Transplanted aNPCs localize to the perinatal white matter, incorporating into the CC, fimbria of the hippocampus, and periventricular parenchyma. A, Postinjection, aNPCs anatomically incorporate into major white matter tracts of P0-injected animals. B–D, Cells are present in the CC (B), hippocampal fimbria (C), and periventricular parenchyma (D).
Figure 2.
Figure 2.
Transplanted stem cell fate favors oligodendrocytic lineage and cells associate with axons in the CC. A, E, I, YFP+ NPCs extend processes that associate with NF200+ axons in the CC. B, C, F, G, J, K, N, R, V, AA, Complete eYFP colocalization is present with OL markers MBP (B, F, J, late myelin marker), MAG (N, R, V; in extruded processes, mid-developmental marker), and Olig2 (AA, early lineage marker), while MBP immunoreactive projections only interact with the NF200+ axons (C, G, K). M, O, Q, S, U, W, Conversely, eYFP (M, Q, U) and MAG (O, S, W) expression does not overlap with astrocytic marker GFAP. D, H, L, P, T, X, AA, Merged images. Y, Z, Singly stained eYFP (Y) and Olig2 (Z) immunopositivity. AB, Quantification of neuronal marker NeuN+, GFAP+, and Olig2+ cells shows 96.5 ± 0.6% oligodendroglial differentiation, with 1.3 ± 0.6% and 2.0 ± 0.4% neuronal and astroglial differentiation respectively (n = 9). Data are presented as means ± SEM.
Figure 3.
Figure 3.
Transplanted cells incorporate in an age-dependent manner. A–L, Cell incorporation following transplant into neonatal P0 (A–D), P7 (E–H), and P21 (I–L) animals. A, E, I, Gross incorporation of YFP+ cells, after injection at P0, P7, and P21 respectively. B, F, J, Presence of YFP+ cells. C, G, K, Evidence that YFP+ cells can express MBP [P0-injected (C), P7-injected (G), and P21-injected (K) animals], the gene lacking in the Shiverer mutation. D, H, L, Merged images. M, Quantification shows age-dependent linearity in area of YFP+ incorporation. P0, n = 3; P7, n = 4, P21, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 respectively, in one-way ANOVA, followed by Tukey's post hoc test. Data are presented as means ± SEM.
Figure 4.
Figure 4.
Newly formed myelin enwraps axons, creating regions with dense, dark, compact myelin and enabling CAP prediction using computer models. MBP gene product is absent in shiverer mice but produced by transplanted cells. A, Merged image of transplanted eYFP+ stem cells (green) expressing MBP (red). B, Orthogonal section of eYFP/MBP+ cell processes, which interact with NF200+ axons. C, TEM sections of transplanted dysmyelinated shiverer animals, which were preselected for eYFP+ cell presence, showed myelin thickness reaching ∼200 nm. D, G, Normal WT myelin compacts around axons, forming multilamellar structures. E, H, Shiverer myelin congenitally lacks this. F, I, In transplanted shiverer animals, aNPCs enwrapped axons, restoring a multilamellar myelination banding pattern. J, Bands were quantified and a frequency histogram was created for all axons with ≥7 lamellae. K, Transplantation in shi/shi animals restored an intermediate phenotype in the percentage of myelinated cells with at least seven bands. L, Based on TEM lamellar quantification, published literature, and histological measurements (above, Table 1), a computer model of action potential properties was generated to gauge stimulation, recorded by a measuring electrode. M, Unilamellar myelin is predicted to have a CAP speed of 0.62 m/s, with multilamellar myelin producing a signal with approximately double unilamellar CV. N = 3 per group. ***p < 0.001 in one-way ANOVA, followed by Tukey's post hoc test. Data are presented as means ± SEM.
Figure 5.
Figure 5.
Transplanted aNPC-derived myelin normalizes ion channel profiling. A, B, Caspr-mediated compact myelin attachment is present in WT (A) but lost in shi/shi (B) mice. C, D, aNPC transplantation results in significantly more compact Caspr, reverting it to WT phenotype. E, F, Voltage-gated Na+ channels (Nav1.6) are normally distributed in nodes of Ranvier (E), but are disturbed in shi/shi mice (F). G, H, aNPC transplantation does not affect Na+ channel distribution and number of nodal structures quantified by Nav1.6-Caspr double-staining. I, J, Distribution of voltage-gated K+ channels (Kv1.2), normally present in juxtaparanodal regions (I), is disturbed in shi/shi mice (J). K, aNPC transplantation reorganizes Kv1.2 to its native juxtaparanodal region. L, Quantification of nodal structures using Kv1.2-Caspr double-staining shows significant increase in number of nodes after aNPC transplantation (N = 3 per group). M, Pharmacological assessment of voltage-dependent K channels in shi/shi CC after aNPC transplantation shows that 4-AP can enhance CAP in the slow N2 component in the WT axons, but not in the fast N1 component. It can enhance the N2 component in the shi/shi animals. N, It can enhance both N1 and N2 components in the aNPC-transplanted animals, described in statistical summary (N = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001 respectively, in one-way ANOVA, followed by Tukey's post hoc test. Data are presented as means ± SEM.
Figure 6.
Figure 6.
Electrophysiological evidence of myelination and enhancement in axonal conductance after aNPC transplantation into shiverer CC. A, Dual recording of CAPs from the CC in shiverer mouse brain slices. Positions of the stimulation electrode and two recording electrodes are shown. B, Bimodal signal velocity is restored in transplanted shi/shi animals to normal levels. C, Dual-channel recording reveals single-peak CAPs in the shiverer CC, but double peaks in P21 aNPC-transplanted shi/shi (shi-shi + aNPCs) CC, comparable to double peaks visible in WT sections (Crawford et al., 2009a). D, CAPs mediated by both newly myelinated and dysmyelinated axons in the aNPC-transplanted shi/shi CC could be blocked by application of sodium channel blocker TTX (1.0 μm) for ∼10 min. E, Estimated CV using linear regression from multiple shi/shi slices (N = 7) and aNPC-transplanted shi/shi slices (N = 10) reveals restoration of CAPs with accumulated velocities comparable to model predictions. ***p < 0.001 in one-way ANOVA, followed by Tukey's post hoc test. Data are presented as means ± SEM.
Figure 7.
Figure 7.
Myelin reveals normalized axonal profile: activation threshold, refractoriness, and HFS. A, Myelination lowers the threshold for axon activation. CAPs recorded under different stimulation intensities in the WT, shi/shi, and aNPC-transplanted (shi/shi + aNPCs) CC. Separate traces from A show that the threshold for activation of the fast, myelinated axons (N1) is lower than that for the slow, native dysmyelinated axons (N2) in the aNPC-transplanted CC (n = 6). B, I–O curves for WT (N1 and N2 components), shi/shi (N2 component), and aNPC-transplanted shi-shi animals (N1 and N2 components). The minimal current that can elicit observable CAP is 0.2 mA for the fast N1 component in both the WT and aNPC-transplanted shi/shi animals, but is ∼0.3 mA for the slow N2 component in all three animal groups. There is no statistically significant difference in the activation threshold between the slow component (N2) in the aNPC-transplanted CC and that in shi/shi animals (p > 0.05). In aNPC-transplanted shi/shi, the N1 component shows lower activation threshold than the N2 component. *p < 0.05 at several low stimulation intensities. C, New myelination reduces axonal refractoriness in the shi/shi CC. Superimposed CAPs evoked by paired stimuli of varying intervals from an aNPC-transplanted slice. The fast, newly myelinated axons (N1) have less refractoriness than the slow, dysmyelinated axons (N2), measured by absolute refractory period during pair-pulse stimulation. D, Single traces, as in A, with different pair-pulse intervals. E, Statistical comparison of the ratio of CAP2/CAP1 values in the N1 and N2 components in the CAPs recorded from the aNPC-transplanted axons (N = 6). F, Myelination enhances the response of the axons to HFS. Shi/shi CC under 20 Hz, 5 s stimulation. The CAP peak disappeared after ∼5 s stimulation. The second trace in F shows transplanted CC under 20 Hz, 5 s stimulation. Note the slow peak (N2) disappeared during stimulation while the fast peak (N1) persisted. G, Statistical summary comparing results from shi/shi (N = 6) and aNPC-transplanted shi/shi (shi/shi + aNPC) slices (n = 6). Myelinated axons have much improved capability to propagate action potentials under HFS than native, dysmyelinated axons in aNPC-transplanted CC. No significant difference was observed between the dysmyelinated axons in the shi/shi CC and those in aNPC-transplanted animals. *p < 0.05 and **p < 0.001 respectively, in paired t test or one-way ANOVA, as appropriate. Data are presented as means ± SEM.
Figure 8.
Figure 8.
Myelination enhances ischemic endurance. A, Representative CAP traces recorded from the WT CC, from shi/shi CC, and from the aNPC-transplanted CC during OGD. Note that after 15 min OGD challenge, CAP mediated by the native, dysmyelinated axons (N2) in the aNPC-transplanted CC disappeared. In contrast, a fairly large fast peak (N1), mediated by newly myelinated axons, persisted, although its amplitude was significantly decreased by OGD challenge. B, Statistical summary of N1 and N2 amplitude changes in OGD for WT CC (n = 6), shi/shi CC (n = 6), and aNPC-transplanted shi-shi (shi/shi + aNPC) CC (n = 6). *p < 0.05, ***p < 0.001 in one-way ANOVA, followed by Tukey's post hoc test. Data are presented as means ± SEM.

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