Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4

Journal name:
Nature
Volume:
497,
Pages:
369–373
Date published:
DOI:
doi:10.1038/nature12069
Received
Accepted
Published online

Postnatal/adult neural stem cells (NSCs) within the rodent subventricular zone (SVZ; also called subependymal zone) generate doublecortin (Dcx)+ neuroblasts that migrate and integrate into olfactory bulb circuitry1, 2. Continuous production of neuroblasts is controlled by the SVZ microenvironmental niche3, 4. It is generally thought that enhancing the neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear whether there are conditions that favour astrogenesis over neurogenesis in the SVZ niche, and whether astrocytes produced there have different properties compared with astrocytes produced elsewhere in the brain5. Here we show in mice that SVZ-generated astrocytes express high levels of thrombospondin 4 (Thbs4)6, 7, a secreted homopentameric glycoprotein, in contrast to cortical astrocytes, which express low levels of Thbs4. We found that localized photothrombotic/ischaemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-creERtm4 lineage tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not Dcx+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production8. Consequently, Thbs4 homozygous knockout mice (Thbs4KO/KO) showed severe defects in cortical-injury-induced SVZ astrogenesis, instead producing cells expressing Dcx migrating from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular haemorrhage into the brain parenchyma of Thbs4KO/KO mice. Taken together, these findings have important implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members have important roles9, 10.

At a glance

Figures

  1. SVZ generation of Thbs4hi astrocytes.
    Figure 1: SVZ generation of Thbs4hi astrocytes.

    a, Western blot analysis of Thbs4 protein levels in differentiated primary SVZ and cortical (ctx) astrocyte cultures. b, qPCR analyses of Thbs4 levels in FACS-sorted SVZ versus cortical GFP+ astrocytes from GFAP-GFP transgenic mice. *P<0.001, n = 5, Student’s t-test; error bars indicate s.e.m. c, Schematic representation of cortical transplantation strategy. CC, corpus callosum; LV, lateral ventricle. d, Thbs4, tdTomato and GFAP IHC antibody staining of brain sections—2–4weeks after mice were transplanted with lineage-traced primary SVZ NSC cultures derived from tamoxifen-induced nestin-creERtm4; R26R-tdTomato (N4; R26R-tdTomato) mice—showing co-localization between tdTomato, Thbs4 and GFAP (arrowheads). Scale bar: 20μm.

  2. Thbs4hi astrocyte production after photothrombotic cortical injury.
    Figure 2: Thbs4hi astrocyte production after photothrombotic cortical injury.

    a, Schematic representation of photothrombosis injury model, with dashed box indicating region of imaging in b. b, DAB IHC staining for tdTomato in N4; R26R-tdTomato mice induced with tamoxifen, showing a delayed activation of lineage-traced tdTomato+ cells to injury site 14d.p.i. c, Coronal sections of injury site 14d.p.i., IHC stained for GFAP, Thbs4 and tdTomato, showing that lineage-traced tdTomato+ cells adjacent to injury are Thbs4hiGFAP+ astrocytes (arrowheads). d, Quantitative analyses of total tdTomato+ cells at injury site expressing Thbs4 14d.p.i. (88.10±1.99% s.d., n = 3 mice). e, Western blot and quantitative analyses of Thbs4 protein levels in SVZ tissues 3d.p.i. *P<0.001, n = 5, Student’s t-test, error bars indicate s.e.m. f, DAB IHC staining for Thbs4 expression 3d.p.i., Nissl-counterstained. Ipsilateral and contralateral SVZ from the same brain section, imaged under identical conditions. Scale bars: 100μm (b); 20μm (c); 50μm (f).

  3. Notch signalling and regulation of injury-induced SVZ astrogenesis.
    Figure 3: Notch signalling and regulation of injury-induced SVZ astrogenesis.

    a, Photothrombosis cortical injury model, with areas of imaging indicated by dashed boxes. Representative sections 14d.p.i. showing DAB IHC staining for tdTomato from tamoxifen-induced N4; R26R-tdTomato; RBPjkfl/+ (control); N4; R26R-tdTomato; RBPjkKO/fl (RBPjkKO/fl); and N4; R26R-tdTomato; R26R-NICD mice. In control panels, ipsilateral and contralateral hemispheres are shown, corresponding to boxes in diagram (indicated by dashed arrows). b, Quantification of tdTomato+ cells above the corpus callosum in each genetic background 14d.p.i. *P<0.05, n = 6; **P<0.001 (wild type (WT), n = 8; NICD, n = 5), Student’s t-test; error bars indicate s.e.m. c, Western blot analyses of Notch intracellular domain (NICD) protein levels in SVZ tissues collected 3d.p.i., showing upregulation in the ipsilateral over the contralateral side from the same brain. *P<0.005, n = 5, Student’s t-test, error bars indicate s.e.m. d, Differentiation of SVZ adherent neural stem cell cultures, with or without Jag1–Fc, and/or Thbs4 added. e, Western blot analyses comparing Dcx levels after 5days of culture differentiation. f, Quantification of Dcx levels on western blots. *P<0.001, n = 5, Student’s t-test, error bars indicate s.e.m. g, Freshly isolated SVZ tissue 3d.p.i.: immunoprecipitation (IP) with control beads or anti-Notch1 antibody, and blotted with anti-Thbs4 or anti-Notch1 antibodies, detecting Thbs4 pull down (arrow). h, Thbs4 induction of NICD during in vitro differentiation with or without dynasore, 12h after stimulation. i, Western blot analyses of Nfia levels in SVZ tissues collected 3d.p.i., showing upregulation in the ipsilateral over the contralateral side from the same brain. *P<0.005, n = 5, Student’s t-test, error bars indicate s.e.m. Scale bars: 200mm (a); 50μm (d).

  4. SVZ astrogenesis defects in Thbs4 mutant mice after cortical injury.
    Figure 4: SVZ astrogenesis defects in Thbs4 mutant mice after cortical injury.

    a, Western blot analyses of NICD and Nfia protein levels in SVZ tissues collected 3d.p.i. from Thbs4 knockout mice, showing lack of upregulation in the ipsilateral versus contralateral SVZ. b, IHC staining for tdTomato showing Dcx expression from P6 tamoxifen-induced N4; R26R-tdTomato; Thbs4KO/KO mice 14d.p.i. Note robust Dcx+ cells at injury site. CC,corpus callosum (yellow dashed lines); SCJ, striatal–cortical junction. c, Quantification of total tdTomato+ cells around injury site 14d.p.i., co-labelling with GFAP or Dcx. Lack of strong staining for either GFAP or Dcx was marked as (−). *P<0.002, n = 11 mice (control), 5 mice (KO), Wilcoxon rank sum test. d, MRI analyses of littermate pair, Thbs4KO/+ (control) and Thbs4KO/KO (KO) 8d.p.i. First and second panels show SPGR images, horizontal plane, at two echo times (TE, 4.4 and 14.3ms). The third panel shows computed R2* relaxation rate (RR). The fourth panel shows corresponding magnetic susceptibility (MS). Hyperintense magnetic susceptibility indicates area of haemorrhage (Haem., red arrow). Scale bar units in RR = s−1; magnetic susceptibility is in parts per million (p.p.m.). Ctx, cortex; Inj, injury site; OB, olfactory bulb. e, f, Quantitative measurements of mean diffusivity, magnetic susceptibility (Mag. suscep.), comparing contralateral cortex (Contra.) to areas of infarct and oedema caused by injury. Error bars indicate s.d. g, IHC staining of brain sections 7d.p.i. to visualize GFAP+ astrocytes and biotinylated dextran infused through vasculature. Extravascular biotinylated dextran is readily seen around cortical injury site in knockout mice (close-ups from corresponding dashed boxes in Supplementary Fig. 12c). h, Quantification of parenchymal biotinylated dextran fluorescence next to injury site. *P<0.001, n = 5, Student’s t-test; error bars indicate s.e.m. Scale bars: 50μm (b, g).

Videos

  1. Live-imaging of lineage-traced rostral migratory stream Neuroblasts
    Video 1: Live-imaging of lineage-traced rostral migratory stream Neuroblasts
    This movie shows representative live-imaging of tdTomato+ rostral migratory stream (RMS) neuroblasts in acute brain slices, from N4; R26R-tdTomato mice induced with tamoxifen at P6 and imaged 3 weeks later. Left = rostral. Bidirectional travel of RMS neuroblasts in acute slice preparations had been reported previously34. Images were captured once every 5 minutes; total time = 55 minutes.

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Author information

Affiliations

  1. George and Jean Brumley Neonatal-Perinatal Research Institute, Department of Pediatrics, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Eric J. Benner,
    • Rebecca Jo &
    • Chay T. Kuo
  2. Department of Cell Biology, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Dominic Luciano,
    • Rebecca Jo,
    • Khadar Abdi,
    • Patricia Paez-Gonzalez,
    • Cagla Eroglu &
    • Chay T. Kuo
  3. Department of Neurobiology, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Dominic Luciano,
    • Cagla Eroglu &
    • Chay T. Kuo
  4. Department of Anesthesiology, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Huaxin Sheng &
    • David S. Warner
  5. Brain Imaging and Analysis Center, Department of Radiology, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Chunlei Liu
  6. Department of Radiology, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Chunlei Liu
  7. Duke Institute for Brain Sciences, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Cagla Eroglu &
    • Chay T. Kuo
  8. Preston Robert Tisch Brain Tumor Center, Duke University School of Medicine, Durham, North Carolina 27710, USA

    • Chay T. Kuo

Contributions

E.J.B. performed injury and biochemical experiments; D.L. performed gene expression and live-imaging experiments; K.A. performed in vivo immunoprecipitation experiments; P.P.-G. performed SVZ antibody staining and analyses; R.J., H.S. and D.S.W. assisted with injuries and their analyses; C.L. performed MRI scanning and quantitative analyses; C.E. provided reagents and experimental insight; C.T.K. performed transplantations and conceived the project. E.J.B., D.L. and R.J. assembled figures and C.T.K. wrote the paper. All authors discussed results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Video

  1. Video 1: Live-imaging of lineage-traced rostral migratory stream Neuroblasts (6,955 KB, Download)
    This movie shows representative live-imaging of tdTomato+ rostral migratory stream (RMS) neuroblasts in acute brain slices, from N4; R26R-tdTomato mice induced with tamoxifen at P6 and imaged 3 weeks later. Left = rostral. Bidirectional travel of RMS neuroblasts in acute slice preparations had been reported previously34. Images were captured once every 5 minutes; total time = 55 minutes.

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  1. Supplementary Figures (7.2 MB)

    This file contains Supplementary Figures 1-14.

Additional data