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MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin

Nature Cell Biology volume 16, pages 629–638 (2014)Cite this article

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Abstract

The identification of mechanisms that maintain stem cell niche architecture and homeostasis is fundamental to our understanding of tissue renewal and repair. Cell adhesion is a well-characterized mechanism for developmental morphogenetic processes, but its contribution to the dynamic regulation of adult mammalian stem cell niches is still poorly defined. We show that N-cadherin-mediated anchorage of neural stem cells (NSCs) to ependymocytes in the adult murine subependymal zone modulates their quiescence. We further identify MT5-MMP as a membrane-type metalloproteinase responsible for the shedding of the N-cadherin ectodomain in this niche. MT5-MMP is co-expressed with N-cadherin in adult NSCs and ependymocytes and, whereas MT5-MMP-mediated cleavage of N-cadherin is dispensable for the regulation of NSC generation and identity, it is required for proper activation of NSCs under physiological and regenerative conditions. Our results indicate that the proliferative status of stem cells can be dynamically modulated by regulated cleavage of cell adhesion molecules.

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Figure 1: B1 cells in the subependymal zone (SEZ) interact with their niche through N-cadherin homophilic interactions.
Figure 2: Disruption of N-cadherin-mediated adhesion results in the activation of GFAP+ B cells.
Figure 3: MT5-MMP is a feature of adult NSCs.
Figure 4: MT5-MMP activity sheds N-cadherin ectodomain in vivo and modulates NSC proliferation.
Figure 5: MT5-MMP cleaves N-cadherin in adult NSCs.
Figure 6: MT5-MMP activity is necessary for the activation of adult NSCs.
Figure 7: MT5-MMP activity is necessary for the re-activation of adult NSCs in regenerative conditions.

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References

  1. Kriegstein, A. & Alvarez-Buylla, A. The glial nature of embryonic and adult neural stem cells. Annu. Rev. Neurosci. 32, 149–184 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zhao, C., Deng, W. & Gage, F. H. Mechanisms and functional implications of adult neurogenesis. Cell 132, 645–660 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Ferri, A. L. et al. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development 131, 3805–3819 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Maslov, A. Y., Barone, T. A., Plunkett, R. J. & Pruitt, S. C. Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J. Neurosci. 24, 1726–1733 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nam, H. S. & Benezra, R. High levels of Id1 expression define B1 type adult neural stem cells. Cell Stem Cell 5, 515–526 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wilson, A. et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135, 1118–1129 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Doetsch, F., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. Regeneration of a germinal layer in the adult mammalian brain. Proc. Natl Acad. Sci. USA 96, 11619–11624 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pastrana, E., Cheng, L. C. & Doetsch, F. Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny. Proc. Natl Acad. Sci. USA 106, 6387–6392 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Andreu-Agullo, C., Morante-Redolat, J. M., Delgado, A. C. & Farinas, I. Vascular niche factor PEDF modulates Notch-dependent stemness in the adult subependymal zone. Nat. Neurosci. 12, 1514–1523 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Miyatani, S. et al. Neural cadherin: Role in selective cell–cell adhesion. Science 245, 631–635 (1989).

    Article  CAS  PubMed  Google Scholar 

  11. Takeichi, M. Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7, 619–627 (1995).

    Article  CAS  PubMed  Google Scholar 

  12. Marthiens, V., Kazanis, I., Moss, L., Long, K. & Ffrench-Constant, C. Adhesion molecules in the stem cell niche–more than just staying in shape? J. Cell Sci. 123, 1613–1622 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gonzalez-Reyes, A. Stem cells, niches and cadherins: A view from Drosophila. J. Cell Sci. 116, 949–954 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Kiel, M. J., Acar, M., Radice, G. L. & Morrison, S. J. Hematopoietic stem cells do not depend on N-cadherin to regulate their maintenance. Cell Stem Cell 4, 170–179 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kiel, M. J., Radice, G. L. & Morrison, S. J. Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. Cell Stem Cell 1, 204–217 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Li, P. & Zon, L. I. Resolving the controversy about N-cadherin and hematopoietic stem cells. Cell Stem Cell 6, 199–202 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Karpowicz, P. et al. E-Cadherin regulates neural stem cell self-renewal. J. Neurosci. 29, 3885–3896 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Seki, T., Namba, T., Mochizuki, H. & Onodera, M. Clustering, migration, and neurite formation of neural precursor cells in the adult rat hippocampus. J. Comp. Neurol. 502, 275–290 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Shen, Q. et al. Adult SVZ stem cells lie in a vascular niche: A quantitative analysis of niche cell–cell interactions. Cell Stem Cell 3, 289–300 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yagita, Y. et al. N-cadherin mediates interaction between precursor cells in the subventricular zone and regulates further differentiation. J. Neurosci. Res. 87, 3331–3342 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Barnabe-Heider, F. et al. Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation. Nat. Methods 5, 189–196 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Folgueras, A. R. et al. Metalloproteinase MT5-MMP is an essential modulator of neuro-immune interactions in thermal pain stimulation. Proc. Natl Acad. Sci. USA 106, 16451–16456 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Monea, S., Jordan, B. A., Srivastava, S., DeSouza, S. & Ziff, E. B. Membrane localization of membrane type 5 matrix metalloproteinase by AMPA receptor binding protein and cleavage of cadherins. J. Neurosci. 26, 2300–2312 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3, 265–278 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nose, A., Nagafuchi, A. & Takeichi, M. Expressed recombinant cadherins mediate cell sorting in model systems. Cell 54, 993–1001 (1988).

    Article  CAS  PubMed  Google Scholar 

  26. Radice, G.L. et al. Developmental defects in mouse embryos lacking N-cadherin. Dev. Biol. 181, 64–78 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Malatesta, P. et al. Neuronal or glial progeny: Regional differences in radial glia fate. Neuron 37, 751–764 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Consiglio, A. et al. Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors. Proc. Natl Acad. Sci. USA 101, 14835–14840 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Noles, S. R. & Chenn, A. Cadherin inhibition of beta-catenin signaling regulates the proliferation and differentiation of neural precursor cells. Mol. Cell. Neurosci. 35, 549–558 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Nieman, M. T., Kim, J. B., Johnson, K. R. & Wheelock, M. J. Mechanism of extracellular domain-deleted dominant negative cadherins. J. Cell Sci. 112, 1621–1632 (1999).

    CAS  PubMed  Google Scholar 

  31. Fortini, M. E. Gamma-secretase-mediated proteolysis in cell–surface–receptor signalling. Nat. Rev. Mol. Cell Biol. 3, 673–684 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Jaworski, D. M. Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system. Brain Res. 860, 174–177 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Malinverno, M. et al. Synaptic localization and activity of ADAM10 regulate excitatory synapses through N-cadherin cleavage. J. Neurosci. 30, 16343–16355 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Marambaud, P. et al. A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell 114, 635–645 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Reiss, K. et al. ADAM10 cleavage of N-cadherin and regulation of cell–cell adhesion and beta-catenin nuclear signalling. Embo J. 24, 742–752 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sekine-Aizawa, Y. et al. Matrix metalloproteinase (MMP) system in brain: Identification and characterization of brain-specific MMP highly expressed in cerebellum. Eur. J. Neurosci. 13, 935–948 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Uemura, K. et al. Characterization of sequential N-cadherin cleavage by ADAM10 and PS1. Neurosci. Lett. 402, 278–283 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Heuberger, J. & Birchmeier, W. Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb. Perspect. Biol. 2, a002915 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Raponi, E. et al. S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage. Glia 55, 165–177 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Niola, F. et al. Id proteins synchronize stemness and anchorage to the niche of neural stem cells. Nat. Cell Biol. 14, 477–487 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Llano, E. et al. Identification and characterization of human MT5-MMP, a new membrane-bound activator of progelatinase a overexpressed in brain tumors. Cancer Res. 59, 2570–2576 (1999).

    CAS  PubMed  Google Scholar 

  42. Pei, D. Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5-MMP. J. Biol. Chem. 274, 8925–8932 (1999).

    Article  CAS  PubMed  Google Scholar 

  43. Esteve, P. et al. SFRPs act as negative modulators of ADAM10 to regulate retinal neurogenesis. Nat. Neurosci. 14, 562–569 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. Luo, J., Shook, B. A., Daniels, S. B. & Conover, J. C. Subventricular zone-mediated ependymal repair in the adult mammalian brain. J. Neurosci. 28, 3804–3813 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Falcao, A. M. et al. The path from the choroid plexus to the subventricular zone: Go with the flow! Front. Cell. Neurosci. 6, 1–8 (2012).

    Article  Google Scholar 

  46. Shoval, I., Ludwig, A. & Kalcheim, C. Antagonistic roles of full-length N-cadherin and its soluble BMP cleavage product in neural crest delamination. Development 134, 491–501 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Andreyeva, A. et al. C-terminal fragment of N-cadherin accelerates synapse destabilization by amyloid-beta. Brain 135, 2140–2154 (2012).

    Article  PubMed  Google Scholar 

  48. Zhang, L., Yang, X., Yang, S. & Zhang, J. The Wnt/beta-catenin signaling pathway in the adult neurogenesis. Eur. J. Neurosci. 33, 1–8 (2011).

    Article  PubMed  Google Scholar 

  49. Adachi, K. et al. Beta-catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone. Stem Cells 25, 2827–2836 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Piccin, D. & Morshead, C. M. Wnt signaling regulates symmetry of division of neural stem cells in the adult brain and in response to injury. Stem Cells 29, 528–538 (2011).

    Article  CAS  PubMed  Google Scholar 

  51. Ferron, S. R. et al. A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells. Nat. Protocol. 2, 849–859 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Valdés for the initial observation of a neurogenic defect and A. Rodríguez-Folgueras and S. R. Ferrón for critical reading of the manuscript. We also would like to thank M. Malumbres and T. Iglesias for scientific support and comments, A. Chenn (Northwestern University, Chicago, IL, USA) for generously providing the dominant negative Cdh construct and M. J. Palop for help with the mouse colonies, and to acknowledge the support of the Servicios Centrales de Soporte a la Investigación Experimental (UVEG). This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2011-23331 to I.F., SAF2011-30494 to R.K., RyC-2008-02772 and BFU2010-21823 to A.C. and SAF2011-23089 to C.L-O.), from Botín Foundation to C.L-O. and I.F. and from Ministerio de Sanidad y Consumo (CIBERNED and RETIC Tercel) and Generalitat Valenciana (Programa Prometeo, ISIC, and ACOMP) to I.F.

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Authors and Affiliations

  1. Centro de Investigaciones Biomédicas en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28049 Madrid, Spain

    Eva Porlan, Beatriz Martí-Prado, José Manuel Morante-Redolat, Ana C. Delgado, Martina Kirstein & Isabel Fariñas

  2. Departamento de Biología Celular, Universidad de Valencia, 46100 Valencia, Spain

    Eva Porlan, Beatriz Martí-Prado, José Manuel Morante-Redolat, Ana C. Delgado, Martina Kirstein & Isabel Fariñas

  3. Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain

    Eva Porlan

  4. Institut de Biomedicina de la Universitat de Barcelona, (IBUB), 08028 Barcelona, Spain

    Antonella Consiglio

  5. Department of Biomedical Sciences and Biotechnologies, University of Brescia and National Institute of Neuroscience, 25123 Brescia, Italy

    Antonella Consiglio

  6. Cic BioGUNE, Parque Tecnológico de Vizcaya, 48160 Derio, Spain

    Robert Kypta

  7. Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, 33006 Oviedo, Spain

    Carlos López-Otín

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  1. Eva Porlan
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Contributions

All of the authors discussed the experiments and contributed to the text of the manuscript. E.P. conducted most of the experiments and wrote a draft of the manuscript. B.M-P. carried out most of the in vivo stainings and contributed to the maintenance and genotyping of the mouse colonies. E.P., J.M.M-R. and R.K. contributed to the biochemistry, cell-cycle and adhesion experiments. E.P., B.M-P. and A.C. carried out the lentiviral delivery experiments. E.P., B.M-P. and J.M.M-R. carried out the AraC regeneration and antibody infusion experiments. A.C.D. contributed to the whole-mount stainings. C.L-O. generated the Mmp24 mutant mouse line. E.P., J.M.M-R., B.M-P., M.K., C.L-O., R.K. and I.F. analysed the data. I.F. conceived the project, secured funding, supervised the experiments and wrote the manuscript.

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Correspondence to Isabel Fariñas.

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Integrated supplementary information

Supplementary Figure 1 Adult neural stem cells express functional N-cadherin.

(a) Immunofluorescent detection of GFAP (red), β-catenin (blue) and γ-tubulin (green) in the wall of the lateral ventricle of wild type mice. (b) Wholemount staining for N-cadherin (red) and GFP (green) in GFAP–eGFP mice. Note that the GFP+ cell shown is also positive for N-cadherin (white arrowheads). Stainings at specific confocal levels are shown (z). Lower panel; higher magnification of z-stack projection showing a GFP+, B cell. (c) Immunofluorescent detection of the ependymal marker S100β (red), GFP (green) and DAPI (blue) in preparations from co-culture experiments using SEZ homogenates from GFAP–eGFP mice on N-cadherin overexpressing L929 cells. (d) Immunofluorescent detection of GFAP (green), Ki67 (cyan) and N-cadherin (red) in the SEZ of wild type mice. Shown is the staining at a specific confocal level (z). Right set of smaller panels show a higher magnification of the area outlined by the white line in the left panel, at the specified confocal levels. Non-proliferative GFAP+ cells show higher levels of N-cadherin staining (full arrowheads) whilst activated to proliferate, Ki67-GFAP double positive cells, have lowered N-cadherin levels (empty arrowheads). Nuclei are counterstained with DAPI (blue), and the dashed white lines mark the ventricle limit. Scale bars: (a,c,d) 5 μm; (b) 10 μm.

Supplementary Figure 2 N- and E-cadherin expression, and pinwheel structure integrity in GFAP(Cre);Cdh2Δ mice.

(a) Immunostaining for GFAP (red) and N-cadherin (green), and (b) E-cadherin (green) in SEZ coronal sections of adult mice with the conditional deletion of N-cadherin in GFAP+ cells (GFAP(Cre);Cdh2Δ) or control littermates (Cdh2floxed). DAPI was used for counterstaining (blue). The dashed white lines mark the ventricle limit. Note the absence of signal for N-cadherin in the SEZ of conditionally targeted animals, whilst E-cadherin staining remained apparently unaffected. (c) Immunofluorescent staining for the detection of S100β (red) and GFAP (green) in tissue from Cdh2floxed and GFAP(Cre);Cdh2Δ mice, in wholemount preparations and (d) coronal sections. Scale bars: 10 μm.

Supplementary Figure 3 Neurogenic input in GFAP(Cre);Cdh2Δ mice.

(a) Macroscopic images of Cdh2floxed and GFAP(Cre);Cdh2Δ brains. Note the overall increased brain size, and the reduced olfactory bulbs (dashed black lines). (b) Confocal reconstructions of wholemount stainings for the neuroblast marker DCX (gray) in Cdh2floxed and GFAP(Cre);Cdh2Δ mice. (c) Magnified images showing the chain-like structures of the migrating neuroblasts (DCX, orange) at the positions indicated in the schematic representation. Scale bars: (a) 2,500 μm; (b) 200 μm; (c) 50 μm.

Supplementary Figure 4 In vivo localized loss and gain of function assays.

(a,c) Immunofluorescent detection of GFP (green) in wholemounts from intraventricularly infected mice with control vector (Lent-GFP) and dominant-negative cadherin (Lent-GFP-DN-CDH). Infection with Lent-GFP-DN-CDH provokes the detachment of ependymocytes from the ventricle wall. (b,d) Detection of S100β (blue), GFP (green) and EC-Ncadherin (red) in wholemounts of the infected ventricles. Note that the discrete membrane localization of EC-Ncadherin (arrowheads) in GFP+ (asterisks) and non-infected ependymal cells shown in control infections (b) is disrupted in Lent-GFP-DN-CDH infected cells (d). (e) GFAP (green) and Ki67 (red) inmunodetection in the SEZ of DN-CDH intraventricularly infected mice. GFP is shown pseudocolored in orange and nuclei are counterstained with DAPI (blue). (f–h) Immunofluorescent detection of S100β (green) and BrdU (red) in coronal sections from infected mice using control (Lent-GFP, f), dominant-negative cadherin (Lent-GFP-DN-CDH, g) and MT5-MMP (Lent-GFP-MT5-MMP, h) carrying lentiviruses. BrdU-label retaining cells (LRC)-S100β double positive cells are pointed at with white arrowheads, while LRC+ cells negative for S100β are pointed at with empty arrowheads. (i) Staining for GFP (green) and MT5-MMP (red) in coronal sections from control and MT5-MMP infected striata, showing the overexpression of the metalloproteinase in infected cells. Scale bars: (a,c) 200 μm; (b–e) 10 μm; (f–h) 15 μm; (i) 200 μm, 10 μm.

Supplementary Figure 5 In vivo localized loss and gain of function provokes the disruption of pinwheel-structures at the surface of the ventricle.

(a) Immunofluorescent detection of GFP in wholemounts from MT5-MMP infected mice. Note that infection with Lent-GFP-MT5-MMP also provokes the detachment of ependymocytes from the ventricle wall, however the effect is milder than with Lent-GFP-DN-CDH (Supplementary Fig. 4c). (b) Detection of S100β (blue), GFP (green) and EC-Ncadherin (red) in wholemounts of the infected ventricles. Note that the discrete membrane localization of EC-Ncadherin in GFP+ and non-infected (empty arrowheads) ependymal cells is disrupted in Lent-GFP-MT5-MMP infected cells (white arrowheads). Parallel control stainings can be seen in Supplementary Fig. 4b. (c–h) Wholemount staining with anti-GFAP (cyan), β-catenin and γ-tubulin (red) and GFP (green) to assess pinwheel structures (white dashed lines and arrows) in infected ventricles. (c,e,g: general view; d,f,h: detailed images). We were able to find some pinwheels in MT5-MMP-infected ventricles (white arrow), in contrast to DN-CDH infected areas. Scale bars: (a,b) 200 μm; 50 μm; 10 μm. (c,e,g) 10 μm. (d,f,h) 5 μm.

Supplementary Figure 6 Biochemical and cellular characterization of the Mmp24-KO mice.

(a) Upper panel: relative mRNA levels of Cdh2 normalized to the ribosomal protein L32 in semiquantitative RT-PCRs in total RNA extracts of primary neurospheres obtained from Mmp24-KO and wild-type animals and grown for 3 days. Mean ± s.e.m. Two tailed unpaired Student’s t Test. Dots represent extracts from cultures established from independent mice. Statistics source data can be found in Supplementary Table 2. Bottom panel: representative image of the bands quantified. (b) Upper panel: densitometric quantification of β-catenin in total SEZ protein extracts from Mmp24-KO and wild type controls normalized to a loading control. Mean ± s.e.m. Two tailed paired Student’s t Test. Dots represent extracts from cultures established from independent mice. Statistics source data can be found in Supplementary Table 2. Bottom panel: representative immunoblots for β-catenin in total protein extracts of primary neurospheres obtained from Mmp24-KO and wild-type animals and grown for 3 days. β-actin is shown for loading control. (c) Representative immunoblot for N-cadherin, the intracellular active domain of Notch (NICD), and β-actin for loading control in extracts from Mmp24-KO neurospheres lentivirally infected for the expression of FLAG-tagged MT5-MMP or with the empty vector (GFP). (d) FLAG immunoblot showing expression of MT5-MMP in infected cells. See Supplementary Fig. 8 for uncropped western blots. (e) Relative number of primary neurospheres obtained from the SEZ of wild-type and mice deficient in MT5-MMP activity. Mean ± s.e.m. Two tailed unpaired Student’s t Test. Dots represent neurosphere cultures obtained from independent mice. Statistics source data can be found in Supplementary Table 2. (f) Percentage of S100β+ cells within the CldU-LRC+ population in the SEZ of wild-type and Mmp24-KO mice, injected with the nucleotide every 2 h during a 12 h period, and allowed to survive for 28 days before euthanasia. Mean ± s.e.m. Two tailed unpaired Student’s t Test. Each dot represents a mouse. Statistics source data can be found in Supplementary Table 2. (g) Representative images of the immunodetection of S100β positive (arrowheads) and CldU-LRC double positive cells (arrows). Scale bars: 10 μmm.

Supplementary Figure 7 N-cadherin expression in GFAP cells at different time-points after AraC treatment.

(a) Immunofluorescent detection of N-cadherin (green) and GFAP (red) in coronal sections from saline or AraC treated mice. The SEZ is delineated by two white dashed lines. (b) Higher magnification images of insets in (a). In saline controls, N-cadherin labels the membranes of the cells in the ependymal and subependymal areas (dashed lines). Immediately after the removal of the pumps (t = 0 h), the SEZ appears enriched in GFAP cells with high levels of N-cadherin (full arrowheads). Twelve and 24 h after the removal of the treatment, GFAP cells appear to downregulate N-cadherin (arrowheads). Scale bars: (a) 10 μm. (b) 5 μm.

Supplementary Figure 8 Uncropped western blots and DNA agarose gels.

(a,b) Immunoblots (IB) and ponceau-s staining in Fig. 5b, c. (c,d) Immunoblots in Fig. 5e–d. (f–i) Immunoblots and agarose gel in Supplementary Fig. 6a, b, c, d. MW: molecular weight marker; NTC: no template control.

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Porlan, E., Martí-Prado, B., Morante-Redolat, J. et al. MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin. Nat Cell Biol 16, 629–638 (2014). https://doi.org/10.1038/ncb2993

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