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ALK5-dependent TGF-β signaling is a major determinant of late-stage adult neurogenesis

Nature Neuroscience volume 17, pages 943–952 (2014)Cite this article

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Abstract

The transforming growth factor-β (TGF-β) signaling pathway serves critical functions in CNS development, but, apart from its proposed neuroprotective actions, its physiological role in the adult brain is unclear. We observed a prominent activation of TGF-β signaling in the adult dentate gyrus and expression of downstream Smad proteins in this neurogenic zone. Consistent with a function of TGF-β signaling in adult neurogenesis, genetic deletion of the TGF-β receptor ALK5 reduced the number, migration and dendritic arborization of newborn neurons. Conversely, constitutive activation of neuronal ALK5 in forebrain caused a marked increase in these aspects of neurogenesis and was associated with higher expression of c-Fos in newborn neurons and with stronger memory function. Our findings describe an unexpected role for ALK5-dependent TGF-β signaling as a regulator of the late stages of adult hippocampal neurogenesis, which may have implications for changes in neurogenesis during aging and disease.

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Figure 1: Physiological TGF-β signaling is active in postmitotic immature and mature neurons of the adult dentate gyrus.
Figure 2: Ablation of ALK5 results in fewer newborn neurons in the dentate gyrus.
Figure 3: Loss of ALK5 in progenitor cells affects the survival and morphological maturation of newborn neurons in the dentate gyrus.
Figure 4: Overexpression of ALK5CA leads to an increase in the number of newborn neurons in the dentate gyrus.
Figure 5: Overexpression of ALK5CA accelerates migration and dendritic development of newborn neurons in the adult dentate gyrus.
Figure 6: Overexpression of ALK5CA results in the expression of hippocampal genes involved in JNK signaling.
Figure 7: Overexpression of ALK5CA enhances activity of newborn neurons in the dentate gyrus and improves memory of ALK5CA mice.

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References

  1. Krieglstein, K., Zheng, F., Unsicker, K. & Alzheimer, C. More than being protective: functional roles for TGF-beta/activin signaling pathways at central synapses. Trends Neurosci. 34, 421–429 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Blobe, G.C., Schiemann, W.P. & Lodish, H.F. Role of transforming growth factor beta in human disease. N. Engl. J. Med. 342, 1350–1358 (2000).

    CAS  PubMed  Google Scholar 

  3. Unsicker, K., Flanders, K.C., Cissel, D.S., Lafyatis, R. & Sporn, M.B. Transforming growth factor beta isoforms in the adult rat central and peripheral nervous system. Neuroscience 44, 613–625 (1991).

    CAS  PubMed  Google Scholar 

  4. Shi, Y. & Massagué, J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685–700 (2003).

    CAS  PubMed  Google Scholar 

  5. Feng, X.H. & Derynck, R. Specificity and versatility in TGF-β signaling through Smads. Annu. Rev. Cell Dev. Biol. 21, 659–693 (2005).

    CAS  PubMed  Google Scholar 

  6. Tomoda, T. et al. Transforming growth factor-beta is a survival factor for neonate cortical neurons: coincident expression of type I receptors in developing cerebral cortices. Dev. Biol. 179, 79–90 (1996).

    CAS  PubMed  Google Scholar 

  7. Falk, S. et al. Brain area–specific effect of TGF-beta signaling on Wnt-dependent neural stem cell expansion. Cell Stem Cell 2, 472–483 (2008).

    CAS  PubMed  Google Scholar 

  8. Ishihara, A., Saito, H. & Abe, K. Transforming growth factor-beta 1 and -beta 2 promote neurite sprouting and elongation of cultured rat hippocampal neurons. Brain Res. 639, 21–25 (1994).

    CAS  PubMed  Google Scholar 

  9. Ng, J. TGF-beta signals regulate axonal development through distinct Smad-independent mechanisms. Development 135, 4025–4035 (2008).

    CAS  PubMed  Google Scholar 

  10. Yi, J.J., Barnes, A.P., Hand, R., Polleux, F. & Ehlers, M.D. TGF-beta signaling specifies axons during brain development. Cell 142, 144–157 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Unsicker, K. & Krieglstein, K. TGF-betas and their roles in the regulation of neuron survival. Adv. Exp. Med. Biol. 513, 353–374 (2002).

    CAS  PubMed  Google Scholar 

  12. Tesseur, I. & Wyss-Coray, T. A role for TGF-beta signaling in neurodegeneration: evidence from genetically engineered models. Curr. Alzheimer Res. 3, 505–513 (2006).

    CAS  PubMed  Google Scholar 

  13. Brionne, T.C., Tesseur, I., Masliah, E. & Wyss-Coray, T. Loss of TGF-beta1 leads to increased neuronal cell death and microgliosis in mouse brain. Neuron 40, 1133–1145 (2003).

    CAS  PubMed  Google Scholar 

  14. Tesseur, I. et al. Deficiency in neuronal TGF-beta signaling promotes neurodegeneration and Alzheimer's pathology. J. Clin. Invest. 116, 3060–3069 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Buckwalter, M.S. et al. Chronically increased transforming growth factor-beta1 strongly inhibits hippocampal neurogenesis in aged mice. Am. J. Pathol. 169, 154–164 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Wachs, F.P. et al. Transforming growth factor-beta1 is a negative modulator of adult neurogenesis. J. Neuropathol. Exp. Neurol. 65, 358–370 (2006).

    CAS  PubMed  Google Scholar 

  17. Deng, W., Aimone, J.B. & Gage, F.H. New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat. Rev. Neurosci. 11, 339–350 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Faigle, R. & Song, H. Signaling mechanisms regulating adult neural stem cells and neurogenesis. Biochim. Biophys. Acta 1830, 2435–2448 (2013).

    CAS  PubMed  Google Scholar 

  19. Parent, J.M. Injury-induced neurogenesis in the adult mammalian brain. Neuroscientist 9, 261–272 (2003).

    PubMed  Google Scholar 

  20. Duan, X., Kang, E., Liu, C.Y., Ming, G.L. & Song, H. Development of neural stem cell in the adult brain. Curr. Opin. Neurobiol. 18, 108–115 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Luo, J., Lin, A.H., Masliah, E. & Wyss-Coray, T. Bioluminescence imaging of Smad signaling in living mice shows correlation with excitotoxic neurodegeneration. Proc. Natl. Acad. Sci. USA 103, 18326–18331 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Lin, A.H. et al. Global analysis of Smad2/3-dependent TGF-beta signaling in living mice reveals prominent tissue-specific responses to injury. J. Immunol. 175, 547–554 (2005).

    CAS  PubMed  Google Scholar 

  23. Plümpe, T. et al. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation. BMC Neurosci. 7, 77 (2006).

    PubMed  PubMed Central  Google Scholar 

  24. Mazerbourg, S. & Hsueh, A.J. Genomic analyses facilitate identification of receptors and signaling pathways for growth differentiation factor 9 and related orphan bone morphogenetic protein/growth differentiation factor ligands. Hum. Reprod. Update 12, 373–383 (2006).

    CAS  PubMed  Google Scholar 

  25. Wu, H.H. et al. Autoregulation of neurogenesis by GDF11. Neuron 37, 197–207 (2003).

    CAS  PubMed  Google Scholar 

  26. Muzumdar, M.D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    CAS  PubMed  Google Scholar 

  27. Marxreiter, F. et al. Changes in adult olfactory bulb neurogenesis in mice expressing the A30P mutant form of alpha-synuclein. Eur. J. Neurosci. 29, 879–890 (2009).

    PubMed  Google Scholar 

  28. Imielski, Y. et al. Regrowing the adult brain: NF-kappaB controls functional circuit formation and tissue homeostasis in the dentate gyrus. PLoS ONE 7, e30838 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Mayford, M. et al. Control of memory formation through regulated expression of a CaMKII transgene. Science 274, 1678–1683 (1996).

    CAS  PubMed  Google Scholar 

  30. Jessberger, S., Romer, B., Babu, H. & Kempermann, G. Seizures induce proliferation and dispersion of doublecortin-positive hippocampal progenitor cells. Exp. Neurol. 196, 342–351 (2005).

    CAS  PubMed  Google Scholar 

  31. Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M. & Gage, F.H. Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Development 130, 391–399 (2003).

    CAS  PubMed  Google Scholar 

  32. Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178 (2007).

    CAS  PubMed  Google Scholar 

  33. Hevner, R.F. et al. Tbr1 regulates differentiation of the preplate and layer 6. Neuron 29, 353–366 (2001).

    CAS  PubMed  Google Scholar 

  34. Bury, F.J. et al. Xenopus BTBD6 and its Drosophila homologue lute are required for neuronal development. Dev. Dyn. 237, 3352–3360 (2008).

    CAS  PubMed  Google Scholar 

  35. Resende, R.R., da Costa, J.L., Kihara, A.H., Adhikari, A. & Lorencon, E. Intracellular Ca2+ regulation during neuronal differentiation of murine embryonal carcinoma and mesenchymal stem cells. Stem Cells Dev. 19, 379–394 (2010).

    CAS  PubMed  Google Scholar 

  36. Hirokawa, N., Noda, Y., Tanaka, Y. & Niwa, S. Kinesin superfamily motor proteins and intracellular transport. Nat. Rev. Mol. Cell Biol. 10, 682–696 (2009).

    CAS  PubMed  Google Scholar 

  37. Guzowski, J.F. et al. Mapping behaviorally relevant neural circuits with immediate-early gene expression. Curr. Opin. Neurobiol. 15, 599–606 (2005).

    CAS  PubMed  Google Scholar 

  38. Park, S.M., Jung, J.S., Jang, M.S., Kang, K.S. & Kang, S.K. Transforming growth factor-beta1 regulates the fate of cultured spinal cord–derived neural progenitor cells. Cell Prolif. 41, 248–264 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Prehn, J.H. et al. Regulation of neuronal Bcl2 protein expression and calcium homeostasis by transforming growth factor type beta confers wide-ranging protection on rat hippocampal neurons. Proc. Natl. Acad. Sci. USA 91, 12599–12603 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Krieglstein, K., Suter-Crazzolara, C., Fischer, W.H. & Unsicker, K. TGF-beta superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity. EMBO J. 14, 736–742 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Gleeson, J.G., Lin, P.T., Flanagan, L.A. & Walsh, C.A. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 23, 257–271 (1999).

    CAS  PubMed  Google Scholar 

  42. Gdalyahu, A. et al. DCX, a new mediator of the JNK pathway. EMBO J. 23, 823–832 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Feng, Y. & Walsh, C.A. Protein-protein interactions, cytoskeletal regulation and neuronal migration. Nat. Rev. Neurosci. 2, 408–416 (2001).

    CAS  PubMed  Google Scholar 

  44. Kvajo, M. et al. A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition. Proc. Natl. Acad. Sci. USA 105, 7076–7081 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Enomoto, A. et al. Roles of disrupted-in-schizophrenia 1–interacting protein girdin in postnatal development of the dentate gyrus. Neuron 63, 774–787 (2009).

    CAS  PubMed  Google Scholar 

  46. Duan, X. et al. Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain. Cell 130, 1146–1158 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Porteous, D.J., Thomson, P., Brandon, N.J. & Millar, J.K. The genetics and biology of DISC1–an emerging role in psychosis and cognition. Biol. Psychiatry 60, 123–131 (2006).

    CAS  PubMed  Google Scholar 

  48. Benes, F.M. et al. Regulation of the GABA cell phenotype in hippocampus of schizophrenics and bipolars. Proc. Natl. Acad. Sci. USA 104, 10164–10169 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Marín-Burgin, A. & Schinder, A.F. Requirement of adult-born neurons for hippocampus-dependent learning. Behav. Brain Res. 227, 391–399 (2012).

    PubMed  Google Scholar 

  50. Larsson, J. et al. Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor–deficient mice. EMBO J. 20, 1663–1673 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Wieser, R., Wrana, J.L. & Massague, J. GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex. EMBO J. 14, 2199–2208 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Dittgen, T. et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl. Acad. Sci. USA 101, 18206–18211 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Mosher, K.I. et al. Neural progenitor cells regulate microglia functions and activity. Nat. Neurosci. 15, 1485–1487 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Hughes, R.N. The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory. Neurosci. Biobehav. Rev. 28, 497–505 (2004).

    CAS  PubMed  Google Scholar 

  56. Villeda, S.A. et al. The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature 477, 90–94 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. March, A., Borchelt, D., Golde, T. & Janus, C. Differences in memory development among C57BL/6NCrl, 129S2/SvPasCrl, and FVB/NCrl mice after delay and trace fear conditioning. Comp. Med. 64, 4–12 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Owen, E.H., Logue, S.F., Rasmussen, D.L. & Wehner, J.M. Assessment of learning by the Morris water task and fear conditioning in inbred mouse strains and F1 hybrids: implications of genetic background for single gene mutations and quantitative trait loci analyses. Neuroscience 80, 1087–1099 (1997).

    CAS  PubMed  Google Scholar 

  59. Farley, S.J., McKay, B.M., Disterhoft, J.F. & Weiss, C. Reevaluating hippocampus-dependent learning in FVB/N mice. Behav. Neurosci. 125, 871–878 (2011).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank our laboratory members for discussions and comments, K. Lucin for editorial assistance, J. Luo for comments on data analysis, G. Bieri for providing brain sections of mT/mG mice crossed with Camk2a-cre mice, L. Boerma for helping mouse behavioral tests, P. Osten for providing pFCK(1.3)GW plasmid, and M. Lochrie and the Stanford Neuroscience Gene Vector and Virus Core for generating the viruses (supported by National Institute of Neurological Disorders and Stroke P30 core grant, NS069375-01A1). This work was supported by US National Institutes of Health grant AG27505 (T.W.-C.) and the Department of Veterans Affairs (T.W.-C.).

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  1. Saul A Villeda & Philipp A Jaeger

    Present address: Present address: The Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, USA (S.A.V.), Department of Bioengineering, University of California San Diego, La Jolla, California, USA, and Department of Medicine, University of California San Diego, La Jolla, California, USA (P.A.J.).,

Authors and Affiliations

  1. Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA

    Yingbo He, Hui Zhang, Oluwatobi Olayiwola, Nina Fainberg & Tony Wyss-Coray

  2. Department of Biology, Stanford University, Stanford, California, USA

    Andrea Yung

  3. Center for Tissue Regeneration, Repair and Rehabilitation, VA Palo Alto Health Care System, Palo Alto, California, USA

    Tony Wyss-Coray

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Contributions

Y.H. contributed to all aspects of the experiments and data analysis. H.Z. generated the ALK5CA mice. A.Y. assisted with the Alk5 shRNA experiments and in vivo data analysis. P.A.J. assisted with microarray data analysis. S.A.V. assisted with contextual fear conditioning experiments. O.O. assisted with doxycycline experiments. N.F. assisted with Y maze experiments. Y.H. and T.W.-C. wrote the manuscript. T.W.-C. supervised the project.

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Correspondence to Tony Wyss-Coray.

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Integrated supplementary information

Supplementary Figure 1 Construction and characterization of ALK5cKO transgenic mice.

(a) Diagram of gene constructs used to create ALK5cKO mice. (b,c) Immunoblot of hippocampal lysates from 3-month-old ALK5loxP/loxP and ALK5cKOmice probed with antibodies against ALK5, NSE, and actin in (b) and quantification of ALK5 signal intensity normalized against NSE in (c), n = 2 mice per group, P = 0.0276. Full-length blots are presented in Supplementary Figure 10. The experiments were independently performed twice. (d,e) Representative tiled confocal images (d) and quantification (e) of the dentate gyrus from 3-month-old ALK5loxP/loxP (n = 8) and ALK5cKO(n = 7) mice immunostained for p-Smad2. P-Smad2 signals were normalized to background in (e). Three sections per mouse, P = 0.0433. (f) Representative orthogonal projections from confocal Z-stack images of the dentate gyrus from 3-month-old mT/mG mice before and after crossing with CamK2a-cre mice immunostained for eGFP in combination with Sox2 plus GFAP, MCM2, Tbr2, DCX plus PCNA, and NeuN. DAPI stains nuclei. Scale bar is 100 μm in (d) and 10 μm in (f). (g) Quantification of eGFP positive cells in different cell types detected in (f) from the dentate gyrus. (h) Quantification of eGFP positive and negative cells in individual cell types detected in (f) from the dentate gyrus. n = 4 mice, 3 sections per mouse in (g,h). Data are presented as mean ± s.d. in (c), mean ± s.e.m. in (e,g,h). *P < 0.05, ****P < 0.0001 Student's t-test in (c,e); one-way ANOVA, Dunnett's post-hoc test in (g).

Supplementary Figure 2 Deficiency of ALK5 is not affecting the neural stem cell pool and production of committed neurons.

(a,b) Immunohistochemical detection (a) and quantification (b) of Sox2 labeled cells in the dentate gyrus of 3-month-old ALK5loxP/loxP and ALK5cKO mice (n = 7 mice per group, 5 sections per mouse, P = 0.9023). (c) Quantification of the percentage of BrdU and DCX double-labeled cells in the dentate gyrus from 7-month-old ALK5loxP/loxP and ALK5cKO mice after short-term BrdU labeling (n = 5 mice per group, 5 sections per mouse, P = 0.8043). Experimental design inserted on top. (d) Quantification of total PCNA+ proliferating cells in the dentate gyrus of 3-month-old ALK5loxP/loxP and ALK5cKO mice (n = 7 mice per group, 5 sections per mouse, P = 0.8384) (e) Quantification of the percentage of DCX and PCNA double-labeled cells in the dentate gyrus of 3-month-old ALK5loxP/loxP and ALK5cKO mice (n = 7 mice per group, 5 sections per mouse, P = 0.3450). Scale bar is 100 μm. Data are presented as mean ± s.e.m. Student's t-test.

Supplementary Figure 3 Construction and characterization of cre lentivirus that decreases the survival of infected newborn neurons in ALK5loxP/loxP mouse dentate gyrus.

(a) Design of the lentiviral vectors. (b) Immunohistochemical detection of eGFP labeled lentivirus-infected cells in the dentate gyrus of stereotaxically injected 2-month-old ALK5loxP/loxP mice. (c,d) Representative confocal images (c) and quantification (d) of lentivirus-injected dentate gyrus from 3-month-old ALK5loxP/loxP mice immunostained for p-Smad2 and eGFP. P-Smad2 signals were normalized to signals in uninfected cells in (d). Green or blue dashed circles highlight the cell bodies of eGFP+ or eGFP– neurons. n = 27, 21, 24, 23 neurons from three mice total for eGFP–/LV-eGFP, eGFP+/LV-eGFP, eGFP–/LV-cre-eGFP, eGFP+/LV-cre-eGFP group, respectively. (e) Representative orthogonal projections from confocal Z-stack images of LV-cre-eGFP lentivirus-injected dentate gyrus of2-month-old ALK5loxP/loxP mice immunostained for eGFP in combination with Sox2 plus GFAP, MCM2, Tbr2, DCX plus PCNA, and NeuN. DAPI stains nuclei. Scale bar is 100 μm in (b) and 10 μm in (c,e). (f) Quantification of the number of lentivirus-infected eGFP and DCX double-labeled newborn neurons in the dentate gyrus from LV-cre-eGFP and LV-eGFP stereotaxically injected ALK5loxP/loxP mice with 2 months of age at 6, 14, and 21 dpi. Cell numbers were normalized to values at 6 dpi. n = 4 mice per group, 4 sections per mouse. *P = 0.0355. Data are presented as mean + s.d. in (d) and mean + s.e.m. in (f). ****P < 0.0001. One-way ANOVA, Tukey's post-hoc test (d); one-tailed Student's t-test (f).

Supplementary Figure 4 Construction and characterization of ALK5 shRNA retroviruses that decrease the survival of infected newborn neurons in C57BL/6 mouse dentate gyrus.

(a) Design of the retroviral vectors. MCS, multiple cloning site. (b) Immunoblot of lysates from HEK293T cells co-transfected with an expression vector for ALK5 and different shRNAs probed with antibodies against ALK5 and actin. Full-length blots are presented in Supplementary Figure 10. (c,d) Representative confocal images of retrovirus-injected dentate gyrus of 2-month-old C57BL/6 mice immunostained for eGFP and ALK5 at 7 dpi in (c) or eGFP and p-Smad2 at both 7 dpi and 14 dpi in (d). DAPI stains nuclei. Green or blue dashed circles highlight the cell bodies of eGFP+ or eGFP– neurons. Scale bar is 10 μm. (e) Quantification of p-Smad2 signal intensity from (d). P-Smad2 signals were normalized to signals in uninfected cells in (e). n = 20, 20, 23, 23 neurons from four mice total for eGFP–/shRNA-C, eGFP+/shRNA-C, eGFP–/shRNA-A1, eGFP+/shRNA-A1 group at 7 dpi, respectively. n = 15, 15, 14, 14 neurons from four mice total for eGFP–/shRNA-C, eGFP+/shRNA-C, eGFP–/shRNA-A1, eGFP+/shRNA-A1 group at 14 dpi, respectively. (f) Quantification of the number of virus-infected mCherry only and eGFP-labeled (eGFP only and eGFP/mCherry double-labeled) newborn neurons in the dentate gyrus from stereotaxically injected 2-month-old female C57BL/6 mice at 7 (n = 5 mice), 14 (n = 4 mice, P = 0.0485), and 21 dpi (n = 5 mice, P = 0.0021). Four sections per mouse. Cell numbers were normalized to values at 7 dpi. Data are presented as mean + s.d. in (e) and mean + s.e.m. in (f). *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA, Tukey's post-hoc test (e); Student's t-test (f).

Supplementary Figure 5 Knockdown of ALK5 by shRNA-A2 retrovirus in progenitor cells decelerates dendrite development of newborn neurons in the dentate gyrus.

(a,b) Brightfield immunohistochemical detection of virus-infected eGFP-immunostained cells (left) and confocal images of eGFP, DCX, and DAPI triple-labeled newborn neurons (right) in the dentate gyrus of stereotaxically injected 2-month-old C57BL/6 mice at 7 (a) and 14 dpi (b). Scale bar is 50 μm in brightfield immunohistochemical images and 20 μm in confocal images. (c) Quantification of dendritic length of virus-infected newborn neurons in the dentate gyrus at 7 dpi. (d,e) Quantification of dendritic length (d) and number (e) of virus-infected newborn neurons in the dentate gyrus at 14 dpi. n = 17, 12 neurons from two mice total for each group at 7 dpi. n = 13, 9 neurons from two mice total for each group at 14 dpi. Data are presented as mean + s.d. **P < 0.01, ***P < 0.001, Student's t-test.

Supplementary Figure 6 Construction and characterization of ALK5CA transgenic mice.

(a) Diagram of gene constructs used to create ALK5CA mice. (b) Immunoblots and quantification of p-Smad2 in hippocampal lysates from 3-month-old ALK5Ctrl and ALK5CAmice probed with antibodies against p-Smad2, Smad2, HA, eGFP, and actin. P-Smad2 signal intensity was normalized against Smad2. n = 2 mice per group. Full-length blots are presented in Supplementary Figure 10. Data are presented as mean + s.d. **P = 0.0085, Student's t-test. The experiments were independently performed twice. (c) Representative orthogonal projections from confocal Z-stack images of the dentate gyrus of 4-month-old ALK5CA mice before and after treatment with doxycycline immunostained for eGFP and HA. (d) eGFP expression in the dentate gyrus (dotted line) of ALK5CA mice, but not of ALK5Ctrl mice detected by immunohistochemistry. (e) Representative orthogonal projections from confocal Z-stack images of the dentate gyrus of 3-month-old ALK5CAmice immunostained for eGFP, HA, and DCX. (f) Representative orthogonal projections from confocal Z-stack images of the dentate gyrus of 3-month-old ALK5CAmice immunostained for eGFP in combination with Sox2 plus GFAP, MCM2, Tbr2, DCX plus PCNA, and NeuN. (g) Scheme depicting expression patterns of p-Smad2 and CamK2a-driven genes during neuronal development based on characterization with specific neuronal development markers. DAPI stains nuclei. Scale bar is 100 μm in (d) and 10 μm in (c,e,f).

Supplementary Figure 7 Constitutive activation of ALK5 in forebrain neurons is not affecting neural stem cell pool and production of committed neurons, but is reducing apoptotic markers and increasing newborn neuron survival in the dentate gyrus.

(a) Quantification of Sox2+ cells in the SGZ of the dentate gyrus of 3-month-old ALK5Ctrl and ALK5CA mice (n = 5 mice per group, 5 sections per mouse, P = 0.2514). (b) Experimental design for long-term and short-term BrdU-labeling paradigms. (c) Quantification of the total number of BrdU labeled cells and the percentage of BrdU and DCX double-labeled cells in the dentate gyrus of ALK5Ctrl (n = 7) and ALK5CA (n = 6) mice (3-month-old) 1 day after BrdU administration. Five sections per mouse. (d) A representative confocal image of DAPI staining in the dentate gyrus of 3-month-old ALK5CA mice. Arrow points to an apoptotic cell with a pyknotic nucleus (condensed DAPI staining). Scale bar, 20 μm. (e) Immunoblots of hippocampal lysates from 3-month-old ALK5Ctrl (n = 5) and ALK5CA (n = 4) mice probed with antibodies against Bcl-2, Bax, and NSE. Full-length blots are presented in Supplementary Figure 10. (f) Quantification of BrdU and NeuN double-labeled newborn neurons in the dentate gyrus of 4-month-old ALK5Ctrl and ALK5CA mice 28 days after BrdU administration before and after doxycycline treatment (n = 4 mice per group, 5 sections per mouse). Doxycycline × genotype interaction F(1,12) = 2.783, P = 0.1211; doxycycline treatment F(1,12) = 2.036, P = 0.1791; genotype F(1,12) = 2.134, P = 0.1697; Data are presented as mean ± s.e.m. Student's t-test in (a,c). Two-way ANOVA in (f).

Supplementary Figure 8 Doxycycline treatment suppresses accelerated migration and dendritic development of newborn neurons in ALK5CA mice.

(a) Immunohistochemical detection of DCX labeled neurons in the dentate gyrus (dotted line) from 4-month-old ALK5Ctrl and ALK5CA mice before and after doxycyclline treatment. (b) Relative distribution of DCX labeled cells in ALK5Ctrland ALK5CA mice before and after doxycycline treatment in the ML and three evenly separated layers of the dentate gyrus. (c,d) Dendritic development as quantified by dendritic number (c) and length (d) of DCX labeled cells from ALK5Ctrland ALK5CA mice before and after doxycycline treatment. Scale bar, 50 μm. n = 4 mice per group. In (c), doxycycline × genotype interaction F(1,12) = 59.12, P < 0.0001; doxycycline treatment F(1,12) = 42.25, P < 0.0001; genotype F(1,12) = 34.91, P < 0.0001. In (d), doxycycline × genotype interaction F(1,12) = 40.28, P < 0.0001; doxycycline treatment F(1,12) = 27.99, P = 0.0002; genotype F(1,12) = 26.15, P = 0.0003. Data are presented as mean + s.e.m. ****P < 0.0001, one-way ANOVA, Tukey's post-hoc test (b); two-way ANOVA, Sidak's post-hoc test (c,d).

Supplementary Figure 9 ALK5CA mice do not show any changes in total number of entries in the Y maze or the percentage of freezing under pre-tone and tone conditions in fear conditioning compared with genotype-matched control mice.

(a) Total number of entries in the Y maze was measured from 3-month-old male WT, tTA, ALK5Ctrl, and ALK5CA mice. n = 18 mice per group. (b) Fear conditioning assessed by the percentage of freezing was displayed by 3-month-old male WT, tTA, ALK5Ctrl, and ALK5CA mice before tone and when re-exposed 24 hours later to the tone. n = 11 mice per group. In (b), time × genotype interaction F(3,40) = 0.1782, P = 0.9106; time F(1,40) = 330.9, P < 0.0001; genotype F(3,40) = 0.1832, P = 0.9072. One-way ANOVA, Tukey's post-hoc test (a); Two-way repeated-measures ANOVA, Bonferroni's post-hoc test (b).

Supplementary Figure 10 Full-length blots presented in figures. The bands of interest are displayed within the dashed boxes.

(a) Full-length blots of Figure 6c. (b) Full-length blots of Supplementary Figure 1b. (c) Full-length blots of Supplementary Figure 4b. (d) Full-length blots of Supplementary Figure 6b. (e) Full-length blots of Supplementary Figure 7e.

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He, Y., Zhang, H., Yung, A. et al. ALK5-dependent TGF-β signaling is a major determinant of late-stage adult neurogenesis. Nat Neurosci 17, 943–952 (2014). https://doi.org/10.1038/nn.3732

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