RESUMO
Cell cycle proteins play essential roles in regulating embryonic and adult neurogenesis in the mammalian brain. A key example is the Retinoblastoma protein (Rb) whose loss disrupts the whole neurogenic program during brain development, but only results in increased progenitor proliferation in the adult subventricular zone (SVZ) and compromised long-term neuronal survival in the adult olfactory bulb (OB). Whether this holds true of neurogenesis in the aged brain remains unknown. In this study, we find no evidence of irregular proliferation or early commitment defects in the mid-aged (12-month-old) and old-aged (20-month-old) SVZ following tamoxifen-inducible Rb knockout (Rb iKO) in mice. However, we highlight a striking defect in early maturation of Rb-deficient migrating neuroblasts along the rostral migratory stream (RMS), followed by massive decline in neuronal generation inside the aged OB. In the absence of Rb, we also show evidence of incomplete cell cycle re-entry (CCE) along with DNA damage in the young OB, while we find a similar trend towards CCE but no clear signs of DNA damage or neurodegenerative signatures (pTau or Synuclein accumulation) in the aged OB. However, such phenotype could be masked by the severe maturation defect reported above in addition to the natural decline in adult neurogenesis with age. Overall, we show that Rb is required to prevent CCE and DNA damage in adult-born OB neurons, hence maintain neuronal survival. Moreover, while loss of Rb alone is insufficient to trigger seeding of neurotoxic species, this study reveals age-dependent non-monotonic dynamics in regulating neurogenesis by Rb.
RESUMO
The use of inducible transgenic Nestin-CreERT2 mice has proved to be an essential research tool for gene targeting and studying the molecular pathways implicated in adult neurogenesis, namely, inside the adult subgranular zone (SGZ) of the dentate gyrus and the adult subventricular zone (SVZ) lining the lateral ventricles. Several lines of Nestin-CreER-expressing mice were generated and used in adult neurogenesis research in the past two decades; however, their suitability for studying neurogenesis in aged mice remains elusive. Here, we assessed the efficiency of Cre-loxP genetic recombination in the aging SVZ using the Nestin-CreERT2/Rosa26YFP line designed by Lagace et al. (J Neurosci 27(46):12623-12629, 2007). This analysis was performed in 12-month-old (middle-aged) mice and 20-month-old (old) mice compared to 2-month-old (young adult) mice. To evaluate successful recombination, our approach relies on the histological assessment of Cre mRNA level of expression and the YFP reporter gene's expression inside the aging SVZ by combining in situ hybridization and immunohistochemistry. Using co-immunolabeling, this approach also provides the advantage of estimating the percentage of recombined progeny [(GFP+Nestin+)/Nestin+] and the rate of cell proliferation [(GFP+Ki67+)/GFP+] inside the aging SVZ niche.
