Running-Activated Neural Stem Cells Enhance Subventricular Neurogenesis and Improve Olfactory Behavior in p21 Knockout Mice

  • Vittoria Nicolis di Robilant
  • Raffaella Scardigli
  • Georgios Strimpakos
  • Felice Tirone
  • Silvia Middei
  • Chiara Scopa
  • Marco De Bardi
  • Luca Battistini
  • Daniele Saraulli
  • Stefano Farioli VecchioliEmail author


In the subventricular zone (SVZ) of the adult brain, the neural stem cells (NSCs) ensure a continuous supply of new neurons to the olfactory bulb (OB), playing a key role in its plasticity and olfactory-related behavior. The activation and expansion of NSCs within the SVZ are finely regulated by environmental and intrinsic factors. Running represents one of the most powerful neurogenic stimuli, although is ineffective in enhancing SVZ neurogenesis. The cell cycle inhibitor p21 is an intrinsic inhibitor of NSCs’ expansion through the maintenance of their quiescence and the restrain of neural progenitor proliferation. In this work, we decided to test whether running unveils the intrinsic neurogenic potential of p21-lacking NSCs. To test this hypothesis, we examined the effect of three different paradigms of voluntary running (5, 12, and 21 days) on SVZ neurogenesis of p21 knockout (KO) male mice at two different stages of development, 2 and 12 months of age. In vivo and in vitro data clearly demonstrate that physical activity is consistent with the activation and expansion of NSCs and with the enhancement of SVZ neurogenesis in p21 KO mice. We also found that 12 days of running contribute to the increase in the number of new neurons functionally active within the OB, which associates with an improvement in olfactory performance strictly dependent on adult SVZ neurogenesis, i.e., the odor detection threshold and short-term olfactory memory. These data suggest that in the adult SVZ of p21 KO mice, NSCs retain a high neurogenic potential, triggered by physical activity, with long-term consequences in olfactory-related behavior.


Adult neurogenesis Subventricular zone Physical activity Cell cycle p21 Olfactory behavior 



This work was supported by Regione Lazio Project FILAS to Stefano Farioli Vecchioli.

Supplementary material

12035_2019_1590_MOESM1_ESM.pdf (3.9 mb)
ESM 1 (PDF 4042 kb)


  1. 1.
    Katsimpardi L, Lledo PM (2018) Regulation of neurogenesis in the adult and aging brain. Curr Opin Neurobiol 53:131–138PubMedCrossRefGoogle Scholar
  2. 2.
    Toda T, Parylak SL, Linker SB, Gage FH (2019) The role of adult hippocampal neurogenesis in brain health and disease. Mol Psychiatry 24(1):67–87PubMedCrossRefGoogle Scholar
  3. 3.
    Anacker C, Luna VM, Stevens GS, Millette A, Shores R, Jimenez JC, Chen B, Hen R (2018) Hippocampal neurogenesis confers stress resilience by inhibiting the ventral dentate gyrus. Nature 559(7712):98–102PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Eisinger BE, Zhao X (2018) Identifying molecular mediators of environmentally enhanced neurogenesis. Cell Tissue Res 371(1):7–21PubMedCrossRefGoogle Scholar
  5. 5.
    Saraulli D, Costanzi M, Mastrorilli V, Farioli-Vecchioli S (2017) The long run: neuroprotective effects of physical exercise on adult neurogenesis from youth to old age. Curr Neuropharmacol 15(4):519–533PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Farioli Vecchioli S, Sacchetti S, Nicolis di Robilant V, Cutuli D (2018) The role of physical exercise and omega-3 fatty acids in depressive illness in the elderly. Curr Neuropharmacol 16(3):308–306PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Hardy D, Saghatelyan A (2017) Different forms of structural plasticity in the adult olfactory bulb. Neurogenesis 4(1):e1301850PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Alonso M, Lepousez G, Sebastien W, Bardy C, Gabellec MM, Torquet N, Lledo PM (2012) Activation of adult-born neurons facilitates learning and memory. Nat Neurosci 15(6):897–904PubMedCrossRefGoogle Scholar
  9. 9.
    Shohayeb B, Diab M, Ahmed M, Ng DHC (2018) Factors that influence adult neurogenesis as potential therapy. Transl Neurodegener 7:4PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Doetsch F, Caillé I, Lim DA, García-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97(6):703–716PubMedCrossRefGoogle Scholar
  11. 11.
    Laywell ED, Rakic P, Kukekov VG, Holland EC, Steindler DA (2000) Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain. Proc Natl Acad Sci U S A 97(25):13883–13888PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Imura T, Kornblum HI, Sofroniew MV (2003) The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP. J Neurosci 23(7):2824–2832PubMedCrossRefGoogle Scholar
  13. 13.
    Urbán N, Guillemot F (2014) Neurogenesis in the embryonic and adult brain: same regulators, different roles. Front Cell Neurosci 27:396Google Scholar
  14. 14.
    Lim DA, Alvarez-Buylla A (2016) The adult ventricular-subventricular zone (V-SVZ) and olfactory bulb (OB) neurogenesis. Cold Spring Harb Perst Biol 8:a018820PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Kosaka K, Aika Y, Toida K, Heizmann CW, Hunziker W, Jacobowitz DM, Nagatsu I, Streit P et al (1995) Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb. Neurosci Res 23(1):73–88PubMedCrossRefGoogle Scholar
  16. 16.
    Saghatelyan A, Carleton A, Lagier S, de Chevigny A, Lledo PM (2003) Local neurons play key roles in the mammalian olfactory bulb. J Physiol Paris 7(4–6):517–528CrossRefGoogle Scholar
  17. 17.
    Kohwi M, Osumi N, Rubenstein JL, Alvarez-Buylla A (2005) Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb. J Neurosci 25(30):6997–7003PubMedCrossRefGoogle Scholar
  18. 18.
    Rochefort C, Gheusi G, Vincent JD, Lledo PM (2002) Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory. J Neurosci 22(7):2679–2689PubMedCrossRefGoogle Scholar
  19. 19.
    Zhao C, Deng W, Gage FH (2008) Mechanisms and functional implications for adult neurogenesis. Cell 132(4):645–660CrossRefGoogle Scholar
  20. 20.
    Llorens-Bobadilla E, Zhao S, Baser A, Saiz-Castro G, Zwadlo K, Martin-Villalba A (2015) Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury. Cell Stem Cell 17(3):329–340PubMedCrossRefGoogle Scholar
  21. 21.
    Otsuki L, Brand AH (2018) Cell cycle heterogeneity directs the timing of neural stem cell activation from quiescence. Science 360(6384):99–102PubMedCrossRefGoogle Scholar
  22. 22.
    Engler A, Rolando C, Giachino C, Saotome I, Erni A, Brien C, Zhang R, Zimber-Strobl U et al (2018) Notch2 signaling maintains NSC quiescence in the murine ventricular-subventricular zone. Cell Rep 22(4):992–1002PubMedCrossRefGoogle Scholar
  23. 23.
    Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13(12):1501–1512CrossRefGoogle Scholar
  24. 24.
    Coqueret O (2003) New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 13(2):65–70PubMedCrossRefGoogle Scholar
  25. 25.
    Gartel AL, Radhakrishnan SK (2005) Lost in transcription: p21 repression, mechanisms, and consequences. Cancer Res 65(10):3980–3985PubMedCrossRefGoogle Scholar
  26. 26.
    Rane CK, Minden A (2014) P21 activated kinases: structure, regulation, and functions. Small GTPases 5:e28003PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Karimian A, Ahmadi Y, Yousefi B (2016) Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair (Amst) 42:63–71CrossRefGoogle Scholar
  28. 28.
    Pechnick RN, Chesnokova V (2009) Adult neurogenesis, cell cycle and drug discovery in psychiatry. Neuropsychopharmacology 34(1):244PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Zonis S, Ljubimov VA, Mahgerefteh M, Pechnick RN, Wawrowsky K, Chesnokova V (2013) p21Cip restrains hippocampal neurogenesis and protects neuronal progenitors from apoptosis during acute systemic inflammation. Hippocampus 23(12):1383–1394PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Farioli-Vecchioli S, Tirone F (2015) Control of the cell cycle in adult neurogenesis and its relation with physical exercise. Brain Plast 1(1):41–54PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Qiu J, Takagi Y, Harada J, Rodrigues N, Moskowitz MA, Scadden D, Cheng T (2004) Regenerative response in ischemic brain restricted by p21cip1/waf1. J Exp Med 199(7):937–945PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Porlan E, Morante-Redolat JM, Marqués-Torrejón MÁ, Andreu-Agulló C, Carneiro C, Gómez-Ibarlucea E, Soto A, Vidal A et al (2013) Transcriptional repression of Bmp2 by p21(Waf1/Cip1) links quiescence to neural stem cell maintenance. Nat Neurosci 16(11):1567–1575PubMedCrossRefGoogle Scholar
  33. 33.
    Marqués-Torrejón MÁ, Porlan E, Banito A, Gómez-Ibarlucea E, Lopez-Contreras AJ, Fernández-Capetillo O, Vidal A, Gil J et al (2013) Cyclin-dependent kinase inhibitor p21 controls adult neural stem cell expansion by regulating Sox2 gene expression. Cell Stem Cell 12(1):88–100PubMedCrossRefGoogle Scholar
  34. 34.
    van Praag H, Kempermann G, Gage FH (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2(3):266–270PubMedCrossRefGoogle Scholar
  35. 35.
    Kempermann G, Fabel K, Ehninger D, Babu H, Leal-Galicia P, Garthe A, Wolf SA (2010) Why and how physical activity promotes experience-induced brain plasticity. Front Neurosci 8:189Google Scholar
  36. 36.
    Vivar C, Potter MC, van Praag H (2013) All about running: synaptic plasticity, growth factors and adult hippocampal neurogenesis. Curr Top Behav Neurosci 15:189–210PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Zhao Z, Sabirzhanov B, Wu J, Faden AI, Stoica BA (2015) Voluntary exercise preconditioning activates multiple antiapoptotic mechanisms and improves neurological recovery after experimental traumatic brain injury. J Neurotrauma 32(17):1347–1360PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Archer T, Fredriksson A (2012) Delayed exercise-induced functional and neurochemical partial restoration following MPTP. Neurotox Res 21(2):210–221PubMedCrossRefGoogle Scholar
  39. 39.
    Piao CS, Stoica BA, Wu J, Sabirzhanov B, Zhao Z, Cabatbat R, Loane DJ, Faden AI (2013) Late exercise reduces neuroinflammation and cognitive dysfunction after traumatic brain injury. Neurobiol Dis 54:252–263PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Brown J, Cooper-Kuhn CM, Kempermann G, Van Praag H, Winkler J, Gage FH, Kuhn HG (2003) Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur J Neurosci 17(10):2042–2046PubMedCrossRefGoogle Scholar
  41. 41.
    Bednarczyk MR, Aumont A, Décary S, Bergeron R, Fernandes KJ (2009) Prolonged voluntary wheel-running stimulates neural precursors in the hippocampus and forebrain of adult CD1 mice. Hippocampus 19(10):913–927PubMedCrossRefGoogle Scholar
  42. 42.
    Blackmore DG, Vukovic J, Waters MJ, Bartlett PF (2012) GH mediates exercise-dependent activation of SVZ neural precursor cells in aged mice. PLoS One 7(11):e49912PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Lee JC, Yau SY, Lee TMC, Lau BW, So KF (2016) Voluntary wheel running reverses the decrease in subventricular zone neurogenesis caused by corticosterone. Cell Transplant 25(11):1979–1986PubMedCrossRefGoogle Scholar
  44. 44.
    Mastrorilli V, Scopa C, Saraulli D, Costanzi M, Scardigli R, Rouault JP, Farioli-Vecchioli S, Tirone F (2017) Physical exercise rescues defective neural stem cells and neurogenesis in the adult subventricular zone of Btg1 knockout mice. Brain Struct Funct 222(6):2855–2876PubMedCrossRefGoogle Scholar
  45. 45.
    Farioli-Vecchioli S, Mattera A, Micheli L, Ceccarelli M, Leonardi L, Saraulli D, Costanzi M, Cestari V et al (2014) Running rescues defective neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells. Stem Cells 32(7):1968–1982PubMedCrossRefGoogle Scholar
  46. 46.
    Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ (1995) Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377(6549):552–557PubMedCrossRefGoogle Scholar
  47. 47.
    Farioli-Vecchioli S, Micheli L, Saraulli D, Ceccarelli M, Cannas S, Scardigli R, Leonardi L, Cinà I et al (2012) Btg1 is required to maintain the pool of stem and progenitor cells of the dentate gyrus and subventricular zone. Front Neurosci 6:124Google Scholar
  48. 48.
    Mignone JL, Kukekov V, Chiang AS, Steindler D, Enikolopov G (2004) Neural stem and progenitor cells in nestin-GFP transgenic mice. J Comp Neurol 469(3):311–324PubMedCrossRefGoogle Scholar
  49. 49.
    Colak D, Mori T, Brill MS, Pfeifer A, Falk S, Deng C, Monteiro R, Mummery C et al (2008) Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells. J Neurosci 28(2):434–446PubMedCrossRefGoogle Scholar
  50. 50.
    Brandt MD, Hübner M, Storch A (2012) Brief report: adult hippocampal precursor cells shorten S-phase and total cell cycle length during neuronal differentiation. Stem Cells 30(12):2843–2847PubMedCrossRefGoogle Scholar
  51. 51.
    Cryan JF, Sweeney FF (2011) The age of anxiety: role of animal models of anxiolytic action in drug discovery. Br J Pharmacol 164(4):1129–1161PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Markel AL, Galaktionov YK, Efimov VM (1989) Factor analysis of rat behavior in an open field test. Neurosci Behav Physiol 19(4):279–286PubMedCrossRefGoogle Scholar
  53. 53.
    Breton-Provencher V, Lemasson M, Peralta MR 3rd, Saghatelyan A (2009) Interneurons produced in adulthood are required for the normal functioning of the olfactory bulb network and for the execution of selected olfactory behaviors. J Neurosci 29(48):15245–15257PubMedCrossRefGoogle Scholar
  54. 54.
    Wang W, Lu S, Li T, Pan YW, Zou J, Abel GM, Xu L, Storm DR et al (2015) Inducible activation of ERK5 MAP kinase enhances adult neurogenesis in the olfactory bulb and improves olfactory function. J Neurosci 35(20):7833–7849PubMedCrossRefGoogle Scholar
  55. 55.
    Kippin TE, Martens DJ, van der Kooy D (2005) p21 loss compromises the relative quiescence of forebrain stem cell proliferation leading to exhaustion of their proliferation capacity. Genes Dev 19(6):756–767PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Sakamoto M, Ieki N, Miyoshi G, Mochimaru D, Miyachi H, Imura T, Yamaguchi M, Fishell G et al (2014) Continuous postnatal neurogenesis contributes to formation of the olfactory bulb neural circuits and flexible olfactory associative learning. J Neurosci 34(17):5788–5799PubMedCrossRefGoogle Scholar
  57. 57.
    Yamaguchi M, Manabe H, Murata K, Mori K (2013) Reorganization of neuronal circuits of the central olfactory system during postprandial sleep. Front Neural Circuits 7:132PubMedPubMedCentralCrossRefGoogle Scholar
  58. 58.
    Veyrac A, Sacquet J, Nguyen V, Marien M, Jourdan F, Didier A (2009) Novelty determines the effects of olfactory enrichment on memory and neurogenesis through noradrenergic mechanisms. Neuropsychopharmacology 34(3):786–795PubMedCrossRefGoogle Scholar
  59. 59.
    Sultan S, Rey N, Sacquet J, Mandairon N, Didier A (2011) Newborn neurons in the olfactory bulb selected for long-term survival through olfactory learning are prematurely suppressed when the olfactory memory is erased. J Neurosci 31(42):14893–14898PubMedCrossRefGoogle Scholar
  60. 60.
    Bragado Alonso S, Reinert JK, Marichal N, Massalini S, Berninger B, Kuner T, Calegari F (2019) An increase in neural stem cells and olfactory bulb adult neurogenesis improves discrimination of highly similar odorants. EMBO J 38(6):e98791PubMedPubMedCentralCrossRefGoogle Scholar
  61. 61.
    Salomoni P, Calegari F (2010) Cell cycle control of mammalian neural stem cells: putting a speed limit on G1. Trends Cell Biol 20(5):233–243PubMedCrossRefGoogle Scholar
  62. 62.
    Lange C, Calegari F (2010) Cdks and cyclins link G1 length and differentiation of embryonic, neural and hematopoietic stem cells. Cell Cycle 9(10):1893–1900PubMedCrossRefGoogle Scholar
  63. 63.
    Artegiani B, Lindemann D, Calegari F (2011) Overexpression of cdk4 and cyclinD1 triggers greater expansion of neural stem cells in the adult mouse brain. J Exp Med 208(5):937–948PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Fasano CA, Dimos JT, Ivanova NB, Lowry N, Lemischka IR, Temple S (2007) shRNA knockdown of Bmi-1reveals a critical role for p21-Rb pathway in NSC self-renewal during development. Cell Stem Cell 1(1):87–99PubMedCrossRefGoogle Scholar
  65. 65.
    Li W, Sun G, Yang S, Qu Q, Nakashima K, Shi Y (2008) Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain. Mol Endocrinol 22(1):56–64CrossRefGoogle Scholar
  66. 66.
    Heldring N, Joseph B, Hermanson O, Kioussi C (2012) Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells. CNS Neurol Disord Drug Targets 11(7):884–892PubMedCrossRefGoogle Scholar
  67. 67.
    Xu H, Wang Z, Jin S, Hao H, Zheng L, Zhou B, Zhang W, Lv H et al (2014) Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression. Biochem Biophys Res Commun 446(1):235–240Google Scholar
  68. 68.
    Sakamoto M, Kageyama R, Imayoshi I (2014) The functional significance of newly born neurons integrated into olfactory bulb circuits. Front Neurosci 8:121PubMedPubMedCentralGoogle Scholar
  69. 69.
    Enwere E, Shingo T, Gregg C, Fujikawa H, Ohta S, Weiss S (2004) Aging results in reduced epidermal growth factor receptor signaling, diminished olfactory neurogenesis, and deficits in fine olfactory discrimination. J Neurosci 24(38):8354–8365PubMedCrossRefGoogle Scholar
  70. 70.
    Pan YW, Kuo CT, Storm DR, Xia Z (2012) Inducible and targeted deletion of the ERK5 MAP kinase in adult neurogenic regions impairs adult neurogenesis in the olfactory bulb and several forms of olfactory behavior. PLoS One 7(11):e49622PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Mastrodonato A, Barbati SA, Leone L, Colussi C, Gironi K, Rinaudo M, Piacentini R, Denny CA et al (2018) Olfactory memory is enhanced in mice exposed to extremely low-frequency electromagnetic fields via Wnt/β-catenin dependent modulation of subventricular zone neurogenesis. Sci Rep 8(1):262Google Scholar
  72. 72.
    Adami R, Pagano J, Colombo M, Platonova N, Recchia D, Chiaramonte R, Bottinelli R, Canepari M et al (2018) Reduction of movement in neurological diseases: effects on neural stem cells characteristics. Front Neurosci 12:336Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Vittoria Nicolis di Robilant
    • 1
  • Raffaella Scardigli
    • 2
  • Georgios Strimpakos
    • 1
  • Felice Tirone
    • 1
  • Silvia Middei
    • 1
  • Chiara Scopa
    • 2
  • Marco De Bardi
    • 3
  • Luca Battistini
    • 3
  • Daniele Saraulli
    • 1
  • Stefano Farioli Vecchioli
    • 1
    Email author
  1. 1.Institute of Cell Biology and NeurobiologyNational Research CouncilMonterotondo (RM)Italy
  2. 2.European Brain Research Institute (EBRI)RomeItaly
  3. 3.Laboratory of NeuroimmunologyFondazione Santa LuciaRomeItaly

Personalised recommendations