Assessment of p130 Transcript and Protein Expression Levels during Adult Neurogenesis in the Absence of Retinoblastoma Protein, pRb

Abstract

Two main sites in the mammalian brain retain adult neurogenesis throughout life: the adult subventricular zone (aSVZ) linked to the olfactory bulb (OB) and the subgranular zone (SGZ) in the dentate gyrus of the hippocampus. Adult neural stem and progenitor cells (aNSPCs) produce specific neuronal subtypes throughout life; however, their rate of division is relatively low, and their differentiation potential is rather limited. The Retinoblastoma protein (pRb) is a major cell cycle protein that regulates neurogenesis in the developing and the adult brain by controlling diverse cellular processes. We have previously shown that conditional deletion of Rb in aSVZ-NSPCs causes an increase in progenitors’ proliferation without affecting stem cells’ self-renewal or differentiation fate. However, loss of Rb severely compromises long-term survival of adult-born neurons in the neurogenic sites. The two other members of the Retinoblastoma family of pockets proteins, p107 and p130, regulate different stages of neural stem and/or progenitor cells (NSPCs) lineage development. While the role of p107 has been examined in the adult brain, p130 is the least studied among all three pocket proteins and its role during adult neurogenesis remains unknown. p130 is primarily expressed in mature neurons and is implicated in maintaining terminal differentiation and long-term survival in cortical neurons in culture. Given that neuronal migration, terminal differentiation and short-term survival in the aSVZ-OB axis are not primarily affected by loss of Rb, we hypothesized that this effect could be attributed, at least partially, to functional compensation by other proteins such as by p130 in this context. As a first step to test the above hypothesis, we assessed and compared p130 mRNA and protein expression levels during adult neurogenesis both in vivo and in vitro in young adult mice carrying an Rb conditional deletion compared with Rb heterozygous controls as well as in cultured cells derived from aSVZ tissues of both genotypes. We crossed Nestin-CreERT2-YFP mice and Rbfloxed/floxed mice to generate the desired genotypes and induced a specific Rb deletion in aNSPCs by tamoxifen treatment. Our results showed a significant increase by 8.5 folds in p130 mRNA level in neurosphere cultures derived from Rb-/- mice compared to Rb+/- controls using qRT PCR. In contrast, Western blot analyses performed on protein lysates extracted from similar neurospheres, and, from dissociated OB tissues following Rb deletion revealed a reduction in p130 protein level, which was more pronounced in neurospheres (n=2 per genotype). These results need further validation of the p130 mRNA level by in situ hybridization, and optimization along with confirmation of the western blot data in vivo. 4 Our current data does not seem to support the presence of functional compensation by p130 in the absence of Rb at least in the context/conditions studied here. However, this conclusion warrants further investigation, which is best carried by examining the direct role(s) of p130 during adult neurogenesis e.g. by inducing its conditional deletion in aNSPCs as well as its combined loss with Rb. The findings of this study and ongoing studies will contribute to our basic understanding of the cellular and molecular processes controlling adult neurogenesis, and thus will have translational implications on how to enhance the regenerative capacity in the adult brain.