Myelinogenic Plasticity of Oligodendrocyte Precursor Cells following Spinal Cord Contusion Injury - PubMed (original) (raw)

. 2017 Sep 6;37(36):8635-8654.

doi: 10.1523/JNEUROSCI.2409-16.2017. Epub 2017 Jul 31.

Greg J Duncan 1 3, Jason R Plemel 4, Michael J Lee 1, Jo A Stratton 4, Sohrab B Manesh 1 2, Jie Liu 1, Leanne M Ramer 5, Shin H Kang 6, Dwight E Bergles 6, Jeff Biernaskie 4 7 8, Wolfram Tetzlaff 9 3 10

Affiliations

Myelinogenic Plasticity of Oligodendrocyte Precursor Cells following Spinal Cord Contusion Injury

Peggy Assinck et al. J Neurosci. 2017.

Abstract

Spontaneous remyelination occurs after spinal cord injury (SCI), but the extent of myelin repair and identity of the cells responsible remain incompletely understood and contentious. We assessed the cellular origin of new myelin by fate mapping platelet-derived growth factor receptor α (PDGFRα), Olig2+, and P0+ cells following contusion SCI in mice. Oligodendrocyte precursor cells (OPCs; PDGFRα+) produced oligodendrocytes responsible for de novo ensheathment of ∼30% of myelinated spinal axons at injury epicenter 3 months after SCI, demonstrating that these resident cells are a major contributor to oligodendrocyte regeneration. OPCs also produced the majority of myelinating Schwann cells in the injured spinal cord; invasion of peripheral myelinating (P0+) Schwann cells made only a limited contribution. These findings reveal that PDGFRα+ cells perform diverse roles in CNS repair, as multipotential progenitors that generate both classes of myelinating cells. This endogenous repair might be exploited as a therapeutic target for CNS trauma and disease.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) leads to profound functional deficits, though substantial numbers of axons often survive. One possible explanation for these deficits is loss of myelin, creating conduction block at the site of injury. SCI leads to oligodendrocyte death and demyelination, and clinical trials have tested glial transplants to promote myelin repair. However, the degree and duration of myelin loss, and the extent and mechanisms of endogenous repair, have been contentious issues. Here, we use genetic fate mapping to demonstrate that spontaneous myelin repair by endogenous oligodendrocyte precursors is much more robust than previously recognized. These findings are relevant to many types of CNS pathology, raising the possibility that CNS precursors could be manipulated to repair myelin in lieu of glial transplantation.

Keywords: NG2 glia; OPCs; Schwann cells; myelin; oligodendrocytes; spinal cord injury.

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Figures

Figure 1.

Figure 1.

Genetic labeling of NG2 glia in tamoxifen-inducible PDGFR_α_CreER uninjured control mice. a, b, After the 2 week tamoxifen washout period, Tamoxifen-induced YFP expression in the cytoplasm was observed in two independent P_DGFR_α_CreER_ mouse lines (I and II) crossed to the YFP reporter line. The majority of PDGFRα+ (red)/Olig2+ (blue) cells exhibited recombination (green) in PDGFR_α_CreER(I):YFP mice (a, a′); recombination was more modest in PDGFR_α_CreER(II):YFP mice (b, b′) when observed in uninjured control mice at 14 d post-tamoxifen treatment. c, d, In both lines, YFP (green), Olig2 (red), and PDGFRα (blue) were coexpressed (c), and the recombined population of cells was also coexpressed with the NG2+ (red) population. Arrows point to rare examples of GFP+ cells not overlapping with NG2+ cell (d). e, PDGFR_α_CreER mice were crossed with membrane-tethered (mGFP; green). f, 3D rendering at 14 d after tamoxifen treatment, the majority of PDGFRα+ cells are recombined (mGFP+; green, arrows) with a small subset of PDGFRα+ cells not recombined (arrowhead). It was rare to find recombined cells that had matured into an oligodendrocyte (CC1+; blue) at time of injury. g, Confocal image including Z-plane from outlined box in f demonstrating that PDGFRα does not overlap with CC1 (blue) cells but it is two independent cells on top of one another. All images were taken in spinal cord cross sections. Scale bars: a, b, e, 200 μm; d, 50 μm; a′, b′, c, f, 10 μm .

Figure 2.

Figure 2.

Recombination in central canal-associated cells, vascular associated cells and a subset of PNS endoneurial cells in PDGFR_α_CreER uninjured control mice. a, b, In the uninjured spinal cord of PDGFR_α_CreER:YFP or mGFP mice, recombination (green) was observed in a subset of blood vessel-associated cells (arrow) located on the outside of the endothelial layer (RECA, red; Glut1, blue) but inside the outer basal lamina, (red; c) consistent with the location of pericytes. The majority of the YFP-expressing vascular-associated cells expressed PDGFRα (blue; d) and PDGFRβ (red; d), referred to previously as type A pericytes. A small subset of the YFP+ vascular associated cells appeared to coexpress of YFP (green) and αSMA (red; e; referred to previously as a type B pericytes marker). f, g, Recombination can also be seen in a small number of cells located peripherally in the wall of the central canal, each with a process extending into the lumen of the canal (f); these cells were also NG2+ (red; g). h–l, A subset of endoneurial cells and pericytes (green) that exhibited recombination (i.e., YFP+ or mGFP+) were found in the dorsal root (h), DRG (i), and sciatic nerve (j) in the uninjured PNS in both lines of PDGFR_α_CreER. Many recombined cells in the dorsal root coexpressed fibronectin (blue; k). Rarely were YFP+ pericyte cell bodies encountered (e.g., arrow, nucleus, 3D rendering; l) with associated processes encircling blood vessels in the dorsal root. m, Importantly, recombined cells in the dorsal root did not coexpress the non-myelinating Schwann cell marker p75 (cyan; m), or the myelinating Schwann cell marker P0 (red; i, j, l, m). All images were taken in spinal cord cross section. Scale bars: a, 100 μm; i, 50 μm; b, c, f, g, h, j, k, m, 10 μm; d, e, l, 5 μm.

Figure 3.

Figure 3.

PDGFRα+ progenitors proliferate and contribute to oligodendrocyte lineage cells in response to SCI. a, Timeline for SCI experiment. Tamoxifen was administered to 8- to 10-week-old mice; mice were given a 2 week tamoxifen washout period before a T9/T10 contusion SCI. Twelve weeks after SCI, recombined YFP+ cells incorporated EdU (red; b) and many recombined cells (green) differentiated into CC1-expressing (red) oligodendrocytes (c, arrow). Note the schematic in top right corner of images indicates approximate location where image was taken based on spinal cord cross section. Using the mGFP reporter mice, a small subset of recombined cells continue to express PDGFRα (blue; d, arrow), whereas the majority of recombined cells are now oligodendrocytes with extended processes ensheathing/myelinating axons and expressing MYRF (red; e, arrow). f, mGFP+ oligodendrocytes expressed the paranodal marker Caspr (arrows) with split channels (f′). There were no differences observed in the overall number of OPCs (PDGFRa+Olig2+) or recombined OPCs (YFP+PDGFRa+Olig2+) across the groups (g). h, The amount of total oligodendrocytes and new YFP+ oligodendrocytes differed significantly among the groups (χ(2)2 = 8.57, p = 0.014 and χ(2)2 = 6.18, p = 0.045, respectively). There was a decrease in the overall oligodendrocytes (CC1+Olig2+) at 3 and 12 wpi compared with the week 12 uninjured group (U(5) = 0.00, p = 0.034 and U(7) = 1.00, p = 0.039, respectively; h). There were more overall oligodendrocytes at 12 wpi compared with 3 wpi (U(8) = 2.00, p = 0.033; h). There were more new oligodendrocytes (YFP+CC1+Olig2+) at 12 wpi compared with the week 12 uninjured group (U(7) = 0.00, p = 0.020; h) but the difference observed between the week 12 uninjured group and 3 wpi did not reach significance (U(5) = 1.00, p = 0.07; h). I, Among the total YFP+Olig2+ population, the percentages of oligodendrocytes (YFP+CC1+Olig2+) and the percentages of OPCs (YFP+PDGFRα+Olig2+) differed significantly among the groups (χ(2)2 = 8.27, p = 0.016; χ(2)2 = 8.27, p = 0.016, respectively). Among the total YFP+Olig2+ population, there was a higher percentage of oligodendrocytes (YFP+CC1+Olig2+) at 12 wpi compared with both 3 wpi (U(8) = 2.00, p = 0.033) and the week 12 uninjured control group (U(7) = 0.00, p = 0.02; i). Reciprocally, there was a lower percentage of OPCs (YFP+PDGFRα+Olig2+) at 12 wpi compared with the 3 wpi (U(8) = 2.00, p = 0.033) and the week 12 uninjured control group (U(7) = 0.00, p = 0.02; i). Images were taken in both epicenter spinal cord cross sections (b–d) and longitudinal sections (e) near epicenter. *p < 0.05; +p < 0.1. Scale bars: b, c, 20 μm; e, 15 μm; d, f, 10 μm; f′, 2 μm. Error bars represent the SEM.

Figure 4.

Figure 4.

Extensive new ensheathment/myelination by oligodendrocytes derived from PDGFRα+ progenitors 12 weeks after SCI. Twelve weeks after contusion injury, large numbers of membrane-bound mGFP+ tubes (green) were observed in PDGFR_α_CreER (II):mGFP mice indicating new ensheathment/myelination (a, b). Slides were stained with antibodies for axons (a, white; b, blue), MBP (a, red; b, purple), mGFP (green), and P0 (data not shown). Note that some images are displayed as flattened images (combining large numbers of _z_-stacks into one image; a, b, b′), whereas others are a single _z_-stack image (a confocal optical section; a′, b″). A proportion of sheaths coexpressed MBP [a′, b″; single optical plane at higher-magnification; arrows denote clear mGFP+ (green) and MBP+ (red or purple) tubes]. c–e, Quantification of axons myelinated by oligodendrocytes after SCI (i.e., MBP+, P0NEG processes) at the lesion epicenter demonstrated group differences (χ(2)2 = 6.37, p = 0.041; c) with significantly less myelinated axons at 3 and 12 wpi compared with uninjured age-matched controls (U(5) = 0.00, p = 0.034 and U(6) = 0.00, p = 0.025, respectively; c). The percentage of newly myelinated axons (surrounded by both mGFP+ and MBP+ tubes) to total myelinated axons (surrounded by just MBP+ tube) differed by group (χ(2)2 = 8.48, p = 0.014) and were significantly higher at 12 wpi compared with 3 wpi and uninjured age-matched controls (U(7) = 0.00, p = 0.014 and U(6) = 0.00, p = 0.025; d). Quantification of MBP expression in mGFP+ sheaths indicative of new myelin (open portion of bar) and of MBPNEG mGFP+ sheaths indicative of either OPC process wrapping or merely ensheathing oligodendrocytes (e, closed portion of bar). Quantification of total mGFP+P0NEG sheaths (combined open and closed portion of bar) differed between groups (χ(2)2 = 6.37, p = 0.041) with significantly more overall mGFP+P0NEG sheaths at 12 wpi compared with uninjured age-matched controls (U(6) = 0.00, p = 0.025; e). Quantification of MBP expression in mGFP+ sheaths indicative of new myelin (open portion of bar) differed between groups (χ(2)2 = 6.37, p = 0.041) with significantly more mGFP+MBP+P0NEG tubes at 12 wpi compared with uninjured age-matched controls (U(6) = 0.00, p = 0.025; e). All images were taken in epicenter spinal cord cross sections. *p < 0.05. Scale bars: b, 200 μm; a, 100 μm; b′, 20 μm; a′, b″, 5 μm. Error bars represent the SEM.

Figure 5.

Figure 5.

PDGFRα+ progenitor-derived Schwann cells express typical hallmarks of Schwann cell myelination in PDGFR_α_CreER:mGFP mice. a, b, Arrows point to paranodal marker Caspr (white; a) and to Schmidt-Lanterman incisures (area where myelin is less compact allowing an accumulation of GFP antibody and a decrease in P0; b, arrows); both indicative of mature nodes of Ranvier and myelination. c, d, 12 weeks after injury, recombined myelinating Schwann cells expressed the transcription factor Krox20 (c, arrow) and were surrounded by a basal lamina (d, arrows point to cell body), both hallmarks of myelinating Schwann cells. All images taken in spinal cord longitudinal sections near epicenter. Scale bars: b–d, 5 μm; a, 3 μm.

Figure 6.

Figure 6.

The majority of myelinating Schwann cells in the injured spinal cord are derived from PDGFRα+ progenitors. a, Twelve weeks after spinal cord contusion in PDGFR_α_CreER:YFP or mGFP mice, P0+ (red) Schwann cell myelin was abundant within the dorsal columns in areas of substantial astrocyte loss. There were two distinct populations (a″) of P0+ myelin sheaths, a YFPNEG population (c′) and a YFP+ population (b, c″); most YFPNEG P0+ myelin sheaths were found closer to the dorsal root entry zone, whereas the YFP+ P0+ sheaths were found mainly medially in the dorsal column (c″). b, Arrowheads point to YFP+/P0+ myelin sheaths with the cell bodies of a Schwann cells in the image plane. The arrow denotes an oligodendrocyte ensheathed YFP+ nerve fiber. The arrowheads denote fibers with a clear one-to-one sheath-to-cell ratio typical for Schwann cells (d, arrowheads). e, Schwann cell myelination was also apparent using the membrane-tethered reporter mGFP and horizontal sections through the lesion site (e, e′; dorsal to the left). f, The number of YFP+ myelinating Schwann cell profiles (green portion of bar) increased between 3 and 12 weeks after SCI (U(12) = 0.00, p = 0.001). There were significantly more overall myelinating Schwann cell profiles (gray + green portion of bar) at 12 wpi compared with 3 wpi (U(12) = 8.00, p = 0.035) and the majority of P0+ tubes were also YFP+. Images were taken in both epicenter spinal cord cross sections (a–d) and longitudinal sections near epicenter (e). *p < 0.05. Scale bars: a, e, 200 μm; c′, c″, a′, a″, 20 μm; e′, 10 μm; b, d, 5 μm. Error bars represent the SEM.

Figure 7.

Figure 7.

Olig2+ cells give rise to myelinating Schwann cells after SCI. Olig2creER:YFP mice were used to fate-map oligodendroglial lineage cells. a, In the uninjured thoracic spinal cord, recombination (YFP; green) occurred in Olig2+ cells (white) across the oligodendrocyte lineage [preferentially observed in CC1+ oligodendrocytes (red) and to a lesser extent PDGFRα+ OPCs (blue)] and a subset of gray matter GFAP+ astrocytes. b, There was no recombination observed in cells associated with the central canal [blue outline surrounds central canal; split channels with axons (white) and YFP (green); b″] or the PNS (dorsal root; b‴). c, 12 weeks after SCI, YFP+ cells were abundant at the lesion epicenter and a subset of the YFP+ cells demonstrated typical Schwann cell markers and morphology in the dorsal columns (c′, c″) and in close proximity to a cavitation at the epicenter (d, d′). All images were taken in spinal cord cross sections. Scale bars: a–c, 200 μm; d, 50 μm; a′, 20 μm; b′, b″, b‴, c′, c″, d′, 10 μm.

Figure 8.

Figure 8.

PDGFRα+ cells from the adult DRG and spinal root of PDGFR_α:H2BGFP_ mice do not exhibit Schwann cell fate in vitro. Sciatic nerve (a) and DRG/spinal root (b) -derived cell suspensions from adult PDGFR_α:H2BGFP_ mice were sorted for GFP-expression by FACS. c–f, PDGFRα:H2BGFP+ and PDGFRα:H2BGFPNEG cells were grown in Schwann cell proliferation/differentiation media for 1 week. The bipolar morphology of PDGFRαH2BGFPNEG cells was consistent with Schwann cell differentiation (d, f, arrow). PDGFRαH2BGFP+ cells derived from both peripheral sources exhibited a flattened morphology under the same conditions. g–n, Consistent with morphological findings, PDGFRαH2BGFP+ cells derived from the sciatic nerve (g, i, k, m) or DRG/roots (h, j, l, n) did not express Schwann cell lineage markers such as p75 (g, h), nestin (i, j), and Sox2 (k, l) but expressed αSMA, consistent with a fibroblast-like phenotype. o–v, In contrast, isolated PDGFRα:H2BGFPNEG cells derived from the sciatic nerve (o, q, s, u) or DRG/roots (p, r, t, v) expressed markers of Schwann cell precursors such as p75 (o, p), nestin (q, r), and Sox2 (s, t). Some αSMANEG cells were found in the GFPNEG fraction (u, v, arrows). Scale bars: c–f, 100 μm; g–v, 20 μm.

Figure 9.

Figure 9.

Recombined PDGFRα+ cells in the PNS do not give rise to P0+ cells in response to peripheral injury. a–c, Four weeks after dorsal root crush (a, b) or sciatic nerve crush injury in PDGFR_α_CreER:mGFP mice (c), mGFP+ cells had branched and flattened processes extending in the endoneurium between clusters of P0+ myelinating Schwann cells. Twelve weeks after a severe dorsal root crush injury (d), there was no evidence of mGFP+/P0+ Schwann cells in the dorsal root (d′, d″) or the DRG (d‴). Only the injured spinal cord harbored recombined cells expressing both mGFP and P0. (d′, arrows). Images were taken in root or sciatic nerve longitudinal sections (a–c) or spinal cord cross sections (d). Scale bars: d, 200 μm; d′, 50 μm; d‴, 20 μm; a–d″,10 μm.

Figure 10.

Figure 10.

P0+ Schwann cells give rise to a small number of P0+ Schwann cells after SCI. In uninjured controls, no recombination was observed within the thoracic spinal cord P0creER:YFP mice (a); recombination (YFP; green) was only observed in the PNS surrounding P0+ tubes consistent with Schwann cell morphology (dorsal root: a′; sciatic nerve: c). Assessment of recombination efficiency in the uninjured roots revealed more YFP+ (green) P0+ (red) tubes in the ventral roots compared with the dorsal roots (d). e, f, Twelve weeks after SCI, YFP+ myelinating Schwann cells were observed at the injury epicenter of P0creERT2:YFP mice treated with tamoxifen 2 weeks before injury (e, e′). The relative contribution of YFP+/P0+ myelin sheaths was low relative to the total number of P0+ myelin sheaths at both 3 and 12 wpi (<5 Schwann cell sheaths per section). There were significantly more overall P0+ profiles (gray + green bar; U(11) = 7.00, p = 0.046) and more P0+/YFPNEG profiles (gray portion of bar; U(11) = 7.00, p = 0.046) at 12 wpi compared with 3 wpi. Images were taken in spinal cord cross sections (a), dorsal root cross sections (b), sciatic nerve longitudinal sections (c), or spinal cord cross sections at epicenter (e). *p < 0.05. Scale bars: a, e, 200 μm; c, 20 μm; a**′**, 10 μm; b, e′, 2 μm. Error bars represent the SEM.

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