Research ArticleNeuroscience

Adult rat myelin enhances axonal outgrowth from neural stem cells

See allHide authors and affiliations

Science Translational Medicine  23 May 2018:
Vol. 10, Issue 442, eaal2563
DOI: 10.1126/scitranslmed.aal2563
  • Fig. 1 Rat NPCs extend greater numbers of axons into spinal cord white matter than gray matter.

    (A) GFP-expressing E14 rat spinal cord–derived NPCs were grafted into sites of C5 spinal cord hemisection lesions (black dotted line indicates graft boundary) 2 weeks after injury. One month after grafting, horizontal sections of spinal cord were immunolabeled for GFP (green) and the neuronal marker NeuN (red). Immunolabeling indicated that rat NPCs extended their axons through spinal cord host white matter (WM) and gray matter (GM). Greater numbers of axons appeared to extend through WM than GM. The rostral section of spinal cord is on the left, and the caudal section is on the right. (B and C) GFP immunolabeling of rat NPC graft–derived axons in transverse sections of rat spinal cord demonstrated larger numbers of axons extending into caudal host spinal cord white matter, three spinal segments below the graft site. (D) Quantification of data in (B) and (C). Mean ± SEM (*P < 0.05, paired, two-tailed t test; n = 4 rats). Scale bars, 500 μm (A), 25 μm (B and C).

  • Fig. 2 Human NSC–derived and rodent NPC–derived neurons preferentially associate with host myelin.

    (A) GFP-labeled human induced pluripotent stem cells (iPSC)–derived NSCs (green) were grafted into sites of C5 hemisection lesions in immunodeficient rats. GFP-expressing graft-derived axons (green/yellow) are shown extending along myelin basic protein (MBP)–immunoreactive intact rat host myelin (red; white arrowheads) and degenerating rat host myelin (black arrowheads). The section shown was taken from six spinal cord segments (T3) below the lesion/graft site. (B) Electron micrograph showing apposition of a rat NPC graft–derived axon from an E14 rat spinal cord–derived NPC graft (black arrowhead) to rat host myelin (white arrowheads). Section was taken two spinal cord segments below a graft placed at a C5 hemisection lesion site. The graft axons are immunogold-labeled for GFP. (Inset) Higher magnification inset showing rat NPC graft–derived axon directly in contact with rat host myelin. (C to E) Transverse sections taken two spinal cord segments caudal to the graft site, showing association of GFP-immunoreactive rat NPC graft–derived axons with rat host myelin. (C) GFP-immunoreactive rat NPC graft–derived axon in contact with rat host myelin. (D) Higher magnification image of (C). (E) GFP-labeled rat NPC graft–derived axon not in direct contact with rat host myelin (F) Percentage of GFP-positive graft-derived axons in direct physical contact with host myelin compared to the probability of direct physical contact assuming random axon growth. Sixty-five percent of graft-derived axons directly contacted rat host myelin. Mean ± SEM (*P < 0.05, Z test; n = 105 axons). Scale bars, 10 μm (A), 0.5 μm (B, C, and E), and 0.2 μm (D).

  • Fig. 3 Human NSC-derived and rat NPC–derived axon growth is stimulated by myelin in vitro.

    (A to D) Adult rat DRG neurons plated on laminin exhibited increased growth, which was inhibited by myelin as expected. This inhibition is quantified in (I) (****P < 0.0001, one-way ANOVA with post hoc Tukey’s test; n = 3 rats; n = 4 wells per rat). (E to H) E14 rat spinal cord–derived NPCs exhibited stimulation of neurite growth on myelin, but not laminin. This growth stimulation is quantified in (J) (****P < 0.0001, one-way ANOVA with post hoc Tukey’s test; n = 9 rat embryos, n = 2 to 3 wells per embryo). Values are normalized to the poly-d-lysine (PDL) substrate for each individual experiment. (K to M) Neurite outgrowth from human iPSC–derived NSCs was stimulated on myelin derived from either monkey or rat spinal cord. A βIII-tubulin label was used to identify axons and is quantified in (N). Values are normalized to the laminin substrate (a required substrate for culturing iPSC-derived NSCs) for each individual experiment (****P < 0.0001, one-way ANOVA, with ***P < 0.001, ****P < 0.0001 post hoc Tukey’s test; n = 3 individual experiments, n = 6 to 10 wells per experiment). Mean ± SEM. Scale bars, 100 μm (A to D), 30 μm (E to H), and 20 μm (K to M). A.U., arbitrary units.

  • Fig. 4 Rat NPCs are inhibited by CSPGs.

    (A and B) Neurite outgrowth from E14 rat spinal cord–derived NPCs was inhibited by CSPG in a dose-dependent manner (***P < 0.001, one-way ANOVA, with *P < 0.05, ***P < 0.001 post hoc Tukey’s test; n = 3 rat spinal cords, n = 2 wells per spinal cord). (C and D) Myelin-dependent neurite outgrowth from rat NPCs derived from rat embryos at different developmental stages was developmentally regulated and declined steadily from E14 to E19 (****P < 0.0001, one-way ANOVA with post hoc Tukey’s test; n = 3 embryos per time point, n = 4 wells per embryo). Values are normalized to the PDL substrate for each individual experiment. Mean ± SEM. Scale bars, 20 μm (A), 30 μm (C).

  • Fig. 5 Myelin activates pERK and reduces cAMP in rat NPCs.

    (A to C) Extracellular signal–regulated kinase (ERK) is activated in cultures of E14 rat spinal cord–derived NPCs grown on PDL, laminin (Lam), or myelin substrates as indicated by (A and B) Western blot (n = 3) and (C) ELISA (*P < 0.05, two-tailed t test; n = 2 ELISAs with n = 3 to 4 wells per condition). (D) Neurite-myelin interactions are mediated by soluble protein ligands; boiling or proteinase K digestion significantly attenuated myelin-mediated activation of neurite outgrowth from E14 rat spinal cord–derived NPCs (n = 3 embryos, n = 3 to 4 wells per embryo). (E) Neurite outgrowth from mouse E12 spinal cord–derived NPCs is not inhibited in the presence of myelin isolated from either Nogo, MAG, or OMgp knockout (KO) mice, or the triple knockout compared to wild-type (WT) myelin (n = 3 to 4 embryos, n = 2 to 3 wells per embryo; ****P < 0.0001, one-way ANOVA, with *P < 0.05, ***P < 0.001, ****P < 0.0001 post hoc Tukey’s test; n.s., not significant). (F) cAMP is reduced upon exposure of E14 rat spinal cord–derived NPCs to myelin as shown by ELISA (*P ≤ 0.05, **P < 0.01, two-tailed t test; n = 2 ELISAs, n = 3 wells per condition). (G) Forskolin (1 mM) administration to increase neuronal cAMP in E14 rat spinal cord–derived NPC cultures resulted in slightly higher neurite outgrowth on laminin, but significantly reduced neurite outgrowth on myelin after 24 hours in vitro (*P < 0.05, **P < 0.01, two-tailed t test; n = 3 embryos, n = 2 to 3 wells per embryo). All values are normalized to the PDL or PDL dimethyl sulfoxide (DMSO) condition for each individual experiment. Mean ± SEM.

  • Fig. 6 RNA-seq of rat NPCs exposed to myelin.

    (A) Shown is the number of differentially expressed transcripts revealed by RNA-seq in mouse E12 spinal cord–derived NPCs cultured on myelin, laminin/myelin, or laminin substrates compared to PDL control substrate [false discovery rate (FDR) < 0.1, n = 3 replicates per condition]. Down-regulated transcripts are shown in magenta and up-regulated transcripts in turquoise. (B) Venn diagram showing uniquely regulated and overlapping transcripts for mouse E12 spinal cord–derived NPCs cultured on myelin substrate (blue), laminin/myelin substrate (green), or laminin substrate alone (brown) compared to PDL control substrate (FDR < 0.1). (C) Heatmap of the 100 most differentially regulated transcripts (FDR < 0.1). Increased expression is shown in red, and reduced expression is shown in green. Transcripts and experimental groups are arranged by hierarchical clustering. Note that experimental groups that contain myelin cluster together. (D) Gene ontology (GO) enrichment analysis of up-regulated transcripts upon myelin stimulation compared to PDL substrate via GOrilla (complete gene list enrichment P value: yellow box = 10−3 to 10−5, orange box = 10−5 to 10−7). (E) Cellular functions of proteins encoded by genes in the gene expression data set assigned using the Ingenuity Pathway analysis software (complete gene list). Predicted activation of cellular function is indicated in turquoise and predicted inhibition in magenta (saturation of color correlates with confidence of prediction).

  • Fig. 7 Negr1 mediates the effects of myelin on mouse NPC neurite outgrowth.

    (A) Negr1 mRNA expression in mouse E12 spinal cord–derived NPCs significantly increased on a myelin substrate compared to a laminin or PDL substrate as shown by RNA-seq (one-way ANOVA with **P < 0.01 post hoc Tukey’s test; n = 3). (B) qPCR shows reduced Negr1 mRNA expression in mouse E12 spinal cord–derived NPCs grown on a myelin substrate as a function of embryonic age (*P < 0.05 two-tailed t test; n = 3 individual experiments). (C) Western blot showing increases in Negr1 protein in mouse E12 spinal cord–derived NPCs after plating on myelin, but not laminin (Lam) or PDL. β-Actin is the loading control. (D) Quantification of Western blot shown in (C) (*P < 0.05 two-tailed t test; n = 5 individual experiments). (E) E12 spinal cord–derived NPCs from Negr1-deficient mice displayed no change in baseline neurite growth observed on a PDL substrate. (F) A 25% reduction in myelin-mediated neurite outgrowth from E12 spinal cord–derived NPCs from Negr1-deficient mice was observed (*P < 0.05 two-tailed t test; n = 5 embryos per genotype, n = 4 wells per embryo. Mean ± SEM for all panels.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/442/eaal2563/DC1

    Fig. S1. NPC-derived axon growth is stimulated by a myelin substrate in vitro.

    Fig. S2. Mouse E12 spinal cord–derived NPC neurite growth is stimulated by a myelin substrate in vitro.

    Fig. S3. Axon (Tau1) growth is stimulated by a myelin substrate in vitro.

    Fig. S4. E14 rat spinal cord–derived NPCs lose their ability to be stimulated by myelin upon in vitro maturation.

    Fig. S5. Growth-dependent mechanisms involving pERK.

    Fig. S6. Quality measures of RNA-seq.

    Fig. S7. qPCR verification of RNA-seq.

    Fig. S8. Overexpression of Negr1 in mature spinal cord–derived NPCs increases neurite growth on a myelin substrate in vitro.

    Table S1. Individual level data for experiments with n < 20.

  • Supplementary Material for:

    Adult rat myelin enhances axonal outgrowth from neural stem cells

    Gunnar H. D. Poplawski, Richard Lie, Matt Hunt, Hiromi Kumamaru, Riki Kawaguchi, Paul Lu, Michael K. E. Schäfer, Grace Woodruff, Jacob Robinson, Philip Canete, Jennifer N. Dulin, Cedric G. Geoffroy, Lutz Menzel, Binhai Zheng, Giovanni Coppola, Mark H. Tuszynski*

    *Corresponding author. Email: mtuszynski{at}ucsd.edu

    Published 23 May 2018, Sci. Transl. Med. 10, eaal2563 (2018)
    DOI: 10.1126/scitranslmed.aal2563

    This PDF file includes:

    • Fig. S1. NPC-derived axon growth is stimulated by a myelin substrate in vitro.
    • Fig. S2. Mouse E12 spinal cord–derived NPC neurite growth is stimulated by a myelin substrate in vitro.
    • Fig. S3. Axon (Tau1) growth is stimulated by a myelin substrate in vitro.
    • Fig. S4. E14 rat spinal cord–derived NPCs lose their ability to be stimulated by myelin upon in vitro maturation.
    • Fig. S5. Growth-dependent mechanisms involving pERK.
    • Fig. S6. Quality measures of RNA-seq.
    • Fig. S7. qPCR verification of RNA-seq.
    • Fig. S8. Overexpression of Negr1 in mature spinal cord–derived NPCs increases neurite growth on a myelin substrate in vitro.
    • Table S1. Individual level data for experiments with n < 20.

    [Download PDF]

Navigate This Article