Role of the Retinoblastoma protein, Rb, during adult neurogenesis in the olfactory bulb

Adult neural stem cells (aNSCs) are relatively quiescent populations that give rise to distinct neuronal subtypes throughout life, yet, at a very low rate and restricted differentiation potential. Thus, identifying the molecular mechanisms that control their cellular expansion is critical for regeneration after brain injury. Loss of the Retinoblastoma protein, Rb, leads to several defects in cell cycle as well as neuronal differentiation and migration during brain development. Here, we investigated the role of Rb during adult neurogenesis in the olfactory bulb (OB) by inducing its temporal deletion in aNSCs and progenitors. Loss of Rb was associated with increased proliferation of adult progenitors in the subventricular zone (SVZ) and the rostral migratory stream (RMS) but did not alter self-renewal of aNSCs or neuroblasts subsequent migration and terminal differentiation. Hence, one month after their birth, Rb-null neuroblasts were able to differentiate into distinct subtypes of GABAergic OB interneurons but were gradually lost after 3 months. Similarly, Rb controlled aNSCs/progenitors proliferation in vitro without affecting their differentiation capacity. This enhanced SVZ/OB neurogenesis associated with loss of Rb was only transient and negatively affected by increased apoptosis indicating a critical requirement for Rb in the long-term survival of adult-born OB interneurons.

Immunohistochemistry Frozen sections were warmed at room temperature -RT-for 30 minutes, then blocked for at least 1 hour in blocking solution [1% BSA (amresco 0332-25G), 0.3% Triton X, 5% goat or donkey serum in 0.1M PBS]. Primary antibodies were applied to sections overnight at RT. The next day, following 3x washes in 1x PBS for 10 minutes each, sections were incubated in secondary antibodies BrdU labeling slides were incubated for 45 seconds in acetone, then washed for 10 min in 1xPBS and incubated in 1N HCl for 20 min at 37°C. Slides were then neutralized in 0.1M Sodium Borate (Na2B4O7; pH=8.5) (Fisher scientific S-249) for 10 min and finally washed with 1x PBS. Antibodies were then applied as described earlier.
Antigen retrieval Antigen retrieval was performed before staining for nuclear proteins including NeuN, Ki67, Sox2 and Dlx2. Slides were treated with fresh 10mM sodium citrate (Fisher scientific BP327-500), pH=6 for 15 min at 95C or, alternatively, incubated in target retrieval solution (Dako, S1700) in similar conditions. Following antigen retrieval, slides were washed in 1x PBS and YFP signal was amplified using TSA (Tyramide Signal Amplification) kit (PerkinElmer-ABC kit Fluorescein SAT701001EA).

In situ hybridization
In situ hybridization on frozen tissue sections using digoxigenin (DIG) labeled RNA probes was performed according to the procedures previously described [1]. Hybridized probes were detected with an AP-conjugated anti-digoxigenin Fab fragment antibody (1:2000, Roche) and visualized with the NBT/BCIP substrate system. Antisense riboprobes for Dlx2 [2], Dlx5 and GAD67 (generous gifts from J. L. Rubenstein, University of California at San Francisco, San Francisco, CA), were prepared from plasmids.
Cell sorting the olfactory bulbs from 5-6 animals carrying the same genotype were dissected and mechanically broken up into small pieces. Tissues were then pooled together and treated with digestion media for 30' at 37C as described above. After digestion, tissues were gently triturated twice and resuspended in DMEM/F12 with 10% FBS to inactivate papain. Then, tissues were triturated again and resuspended in 100%Percoll-PBS buffer solution, for a final concentration of 22% vol./vol. Percoll.
Following 15min centrifugation at 500g, dissociated cells were filtered using 40uM filter to obtain single cells suspension in DMEM-F12 without phenol red. Cells were subsequently processed for cell sorting (BD FACS Aria SORP cell sorter).