Rb-Dependent Regulation of Adult Neurogenesis in the Aging Brain Along the Subventricular Zone – Olfactory Bulb Axis

Abstract

Cell cycle machinery is at the center of maintaining the adult neural stem/progenitor cells (NSPCs) throughout life at the two neurogenic sites: the subgranular zone (SGZ) of the hippocampus, and the subventricular zone (SVZ) whose newborn neurons migrate into the olfactory bulb (OB). The Retinoblastoma protein, Rb, is a critical cell cycle protein which was previously implicated in maintaining neurogenesis in the embryonic and young adult brain. In addition, it was associated with the crucial task of preventing/controlling cell cycle re-entry (CCE) in postmitotic neurons, a process whose failure is indicative of early signs of neurodegeneration. However, the role of Rb in maintaining neurogenesis in the aged brain is still undefined. Here, we study the short-term and long-term effects of an Rb inducible knockout (Rb iKO) mouse model, specifically along the SVZ-OB axis in the mid-aged and old-aged brain. We rely on our established model of NestinCreERT2/Rosa26YFP/Rbflox mice to induce the loss of Rb in a time-dependent manner, through tamoxifen-induced Cre/loxP activation in Nestin-positive brain cells in young and aged mice (Nestin is an intermediate filament known to be expressed in adult NSPCs). We will first address the efficiency of the NestinCreERT2 model in mid-aged (MA, 12-14 months old) and old-aged (OA, 20 months old) brain tissues of mice sacrificed at 28 days post-tamoxifen administration (28dpt), by assessing Cre mRNA expression (through in situ hybridization) together with YFP and Nestin protein expression (by double immunohistochemistry, IHC). Second, we will look at the effect of Rb iKO on NSPC proliferation and neuronal commitment in MA and OA brain tissues, each of mice sacrificed at 28dpt and 60dpt. In addition, we will address neuronal migration along the rostral migratory stream (RMS) and later maturation in the OB in Rb+/- and Rb-/- mice at these timepoints. Finally, we will address CCE and DNA damage profiles in the young-aged (YA)-28dpt and MA-210dpt OB tissues following Rb iKO, along with markers of early neurodegenerative protein aggregations.

We first report a near-complete overlap in Cre mRNA and YFP protein expression in the MA-28dpt and OA-28dpt SVZ, together with a high co-expression of YFP and Nestin in SVZ NSPCs at these timepoints. This indicates that Cre recombination is efficient in our line of choice. Next, we observe no significant variation in co-expression of SVZ YFP with Ki67 (a proliferation marker), DCX (early commitment/differentiation marker), Sox2 (a late stem/early progenitor marker), and pH3 (a late mitotic marker) in Rb+/- and Rb-/- mice at MA-28dpt, MA-60dpt, OA-28dpt and OA-60dpt. Along the RMS, however, we report an impaired neuronal migration following Rb iKO in MA-28dpt, which translates into a significant decline in OB neurogenesis at this timepoint. We then show evidence of incomplete CCE (YFP co-IHC with PCNA, Ki67 and pH3) and DNA damage (YFP co-IHC with γH2A.X) in adult-born OB neurons in Rb-/- mice compared to matching controls at YA-28dpt, but not at MA-210dpt or YA-480dpt. Last, we did not observe an abnormal expression of neurotoxic species (tau and α-synuclein) in the OB at MA-210dpt and YA-480dpt following Rb iKO. In conclusion, we establish the NestinCreERT2 transgenic line as an efficient tool to study acute KO effect in the mid-aged and old-aged mouse brain. We also identify a distinct requirement for Rb during adult neurogenesis in the MA and OA SVZ-OB axis in contrast to the YA brain. Moreover, we emphasize the critical role of Rb in maintaining survival of newborn adult neurons by preventing CCE and accumulation of DNA damage. As a result, this study emphasizes the non-monotonic changes in gene regulation of adult neurogenesis with age, thereby highlighting the limitations and potential of implicating this process in neurorehabilitation and neurodegeneration alike.