Research Articles: Development/Plasticity/Repair Enriched Environment Promotes Adult Hippocampal Neurogenesis through FGFRs https://doi.org/10.1523/JNEUROSCI.2286-20.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.2286-20.2021 Received: 31 August 2020 Revised: 27 December 2020 Accepted: 7 January 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 the authors 1 Enriched environment promotes adult hippocampal neurogenesis through FGFRs 2 3 Abbreviated title: EE promotes neurogenesis via FGFRs 4 5 Marta Grońska-Pęski1,2, J. Tiago Gonçalves1,3, Jean M. Hébert1,2,3* 6 7 1Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 USA 8 2Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 USA 9 3Stem Cell Institute, Albert Einstein College of Medicine, Bronx, NY 10461 USA 10 *corresponding author: [email protected] 11 12 Number of pages: 25 pages 13 Number of figures: 11 14 Number of words: Abstract: 127; Introduction: 626; Discussion: 904. 15 16 Keywords: adult neurogenesis; environmental enrichment; exercise; dentate gyrus; neural stem 17 cells; fibroblast growth factor receptors 18 19 Competing Interests statement: The authors declare no competing interests. 20 21 Acknowledgements 22 We would like to thank Philippe Soriano for his generosity in providing Fgfr reporter mice. We 23 acknowledge support of this research from The Einstein Training Program in Stem Cell 24 Research from the Empire State Stem Cell Fund through New York State Department of Health 25 Contract C34874GG (MGP). NYSTEM C32567GG (JMH), NIH, MH070596 (JMH). 1 26 Abstract 27 The addition of new neurons to existing neural circuits in the adult brain remains of great 28 interest to neurobiology due to its therapeutic implications. The premier model for studying this 29 process has been the hippocampal dentate gyrus in mice, where new neurons are added to 30 mature circuits during adulthood. Notably, external factors such as an enriched environment 31 (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE 32 acts by increasing Fibroblast Growth Factor Receptor (FGFR) function autonomously within 33 neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by 34 EE signal through their mediators, FGFR Receptor Substrate (FRS), to induce stem cell 35 proliferation, and through FRS and PLCγ to increase the number of adult-born neurons, 36 providing a mechanism for how EE promotes adult neurogenesis. 2 37 Significance Statement 38 How the environment we live in affects cognition remains poorly understood. In the 39 current study we explore the mechanism underlying the effects of an enriched environment on 40 the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in 41 forming new memories. A mechanism is provided for how neural precursor cells in the adult 42 mammalian dentate gyrus respond to an enriched environment to increase their neurogenic 43 output. Namely, an enriched environment acts on stem and progenitor cells by activating 44 Fibroblast Growth Factor Receptor signaling through Phospholipase CJ and FGF Receptor 45 Substrate (FRS) proteins to expand the pool of precursor cells. 46 47 Introduction 48 Neurogenesis in adults continues to generate much interest because of its impact on 49 cognition and its ability to be influenced by external factors. The rodent hippocampal dentate 50 gyrus (DG) includes a neurogenic niche, the subgranular zone, where radial glia-like stem cells 51 (RGLs) generate new granule neurons into adulthood (Ming and Song, 2011; Gonçalves et al., 52 2016). Remarkably, neurogenesis in the DG is strongly promoted by an enriched environment 53 (EE) and exercise, which increase proliferation and survival of neurogenic cells and improves 54 cognition (Benarroch, 2013). 55 Brain derived neurothrophic factor (BDNF) signaling has been a leading candidate for 56 mediating at least some of the changes observed in the adult neurogenic lineage in response to 57 environmental factors (Liu and Nusslock, 2018). For example, deletion of the BDNF receptor 58 TRKB in adult neural stem cells blocks the accelerated differentiation of neuroblasts induced by 59 the antidepressant ketamine (Ma et al., 2017). However, while deletion of TRKB during 60 development reduces the effects of exercise on adult neurogenesis (Li et al., 2008), direct 3 61 evidence demonstrating that BDNF-TRKB signaling is required in adult neural stem or 62 progenitor cells to mediate the early expansion of precursors in response to factors such as 63 environmental enrichment and exercise is lacking. BMPs have also been implicated in 64 modulating the impact of environment on increasing the number of new neurons in the adult 65 DG, but in this case by repressing rather than enhancing neurogenesis (Gobeske et al., 2009). 66 The CX3CR1 fractalkine receptor acting in microglia can non-autonomously promote expansion 67 of neural precursors in response to exercise (Vukovic et al., 2012). Yet despite these findings, 68 since the discovery of the positive effects of EE and exercise on adult hippocampal 69 neurogenesis (Kempermann et al., 1997; van Praag et al., 1999), little progress has been made 70 in identifying the molecular pathway(s) operating within early neurogenic precursors to 71 intrinsically promote their expansion in response to external conditions. 72 Although the FGF signaling pathway plays roles throughout developmental 73 neurogenesis, the possibility that FGF2, the most studied FGF ligand, plays a role in adult 74 hippocampal neurogenesis remains unclear (Mudò et al., 2009). Nevertheless, the FGF 75 receptors are necessary and sufficient to promote neurogenesis in the adult DG under standard 76 housing conditions (Kang and Hébert, 2015). Deletion of all three of the expressed Fgfr genes, 77 Fgfr1, Fgfr2, and Fgfr3, in adult stem cells results in a dramatic loss of neurogenesis in the DG. 78 Conversely, and similar to the effects of EE and exercise, increasing FGFR activity in 79 neurogenic cells expands their numbers as well as those of their descendants in the DG (Kang 80 and Hébert, 2015). Despite this requirement for FGFRs in neurogenesis, the role of these 81 receptors in regulating neurogenesis in response to environmental influences is unclear. In 82 support of a possible role, acute stress increases FGF2 expression in astrocytes and 83 hippocampal cell proliferation, while neutralization of FGF2 abolishes this proliferative response 84 (Kirby et al., 2013). Based on these previous findings, we hypothesized that EE acts through 85 FGFRs to enhance adult hippocampal neurogenesis. 4 86 Here, we tested this hypothesis by deleting the 3 Fgfr genes in adult neural precursors 87 and measuring the impact that EE has on neurogenesis in the mutants. As is standard in the 88 field, our paradigm for studying the effects of EE includes voluntary exercise because the mice 89 actively run while exploring their complex surroundings. Hence for simplicity we refer to an 90 enriched environment (EE) as one that includes exercise. We demonstrate that the expansion of 91 neurogenic cells in response to EE depends on increasing FGFR activity. Moreover, we show 92 that FGFR activity operates through FGF receptor substrate (FRS) proteins to expand early 93 neurogenic precursors and through FRS, Phopholipase-CJ and CREB to increase the number 94 of late progenitors. Therefore, this study provides essential new insights into the mechanism by 95 which EE increases hippocampal neurogenesis. 5 96 Materials and Methods 97 98 Animals and tamoxifen administration. All experiments were approved by the Albert Einstein 99 College of Medicine Institutional Animal Care and Use Committee. A maximum of five mice 100 were housed in each cage in a 12–12 light–dark (8 am to 8 pm) facility with free access to water 101 and food. Mice with the Nestin-CreERT2 allele (Balordi and FIshell, 2007) were crossed to mice 102 with conditional floxed Fgfr alleles, Fgfr1fx (Trokovic et al., 2003), Fgfr2fx (Yu et al., 2003), Fgfr3fx 103 (Su et al., 2010), Fgfr1PLCγ (Partanen et al., 1998), and Fgfr1ΔFRS (Hoch and Soriano, 2006), to 104 generate mutants and littermate controls. Fgfr1T2A-H2B-GFP; Fgfr2T2A-H2B-mCherry mice (Molotkov et 105 al., 2017) were used to assess FGFR1 and FGR2 expression in the DG. Both males and 106 females were used and showed no obvious differences; therefore, data for both were pooled. 107 Two-month-old mutants and Cre-negative control littermates were administered an 108 intraperitoneal injection of tamoxifen (5 mg/35 g of body weight) dissolved in corn oil, once a 109 day, every other day for a total of 5 doses, except for Figures 4 and 11C-I where all mice were 110 of the same CreERT2;Fgfr1fx/fx;Fgfr2fx/fx;Fgfr3fx/fx genotype and only animals labeled as Fgfr-3KO 111 mice received tamoxifen, while controls received corn oil. Mice were anesthetized with KetaSet 112 and Xylazine, perfused with 4% paraformaldehyde 5 days, 2-, 3-, or 5-weeks post-tamoxifen for 113 analysis, and post-fixed in 4% PFA overnight. 114 115 BrdU administration. For labeling precursor cells in S-phase, 5-bromo-2'-deoxyuridine (BrdU) 116 (100 mg/kg body weight) was injected intraperitoneally twice daily for 2 or 3 consecutive days 117 before collecting brain samples. For birthdating newly generated neurons, mice were injected 118 with BrdU twice daily for 7 consecutive days starting the day after the last tamoxifen treatment. 119 In no case did mice receiving BrdU exhibit weight loss or other overt signs of BrdU toxicity. 120 6 121 Enriched environment/exercise. Mice were placed in EE for either 10 or 14 days (see 122 timelines in Figure 4).