Background
Neural progenitors in the adult hippocampus continually proliferate and differentiate to the neuronal lineage, and ischemic insult promotes neural progenitors proliferation. However, newborn neurons show a progressive reduction in numbers during the initial few weeks, and the basic mechanisms that control survival of the remaining neurons are not well elucidated. Bcl-2, an anti-apoptotic gene, is a crucial regulator of programmed cell death in CNS development and in apoptotic and necrotic cell death following various stress stimuli, including ischemia. However, the effect of Bcl-2 on adult neurognesis has been presently unknown. Thus, we tested whether Bcl-2 overexpression enhances newborn neurons survival in the adult hippocampus in vivo and in vitro.
Subjects and Methods
Newborn cells profiles: BrdU was used to label proliferating cells. Mice were killed at 1, 7, 14, 21, and 30 days after BrdU administration, transcardially perfused with 4% PFA and immunohistochemistry was performed. Bcl-2 overexpression: To examine the effect of Bcl-2 on new neurons survival, transgenic mice overexpressing human bcl-2 under a NSE promoter (Bcl-2 Tg) were used. Expression of the human-bcl-2 transgene was evaluated, and then the proliferation, differentiation, and survival in the mice were determined. Transient forebrain ischemia: Both common carotid arteries were occluded for 12 min. Thereafter experimental protocol was same as above. Hippocampal primary culture: Hippocampal neuronal cultures with BrdU labeling were prepared from P0 Bcl-2 Tg and their littermates. At 7, 14 and 30 days after seeding, the survivals of newborn neurons were compared between the two groups.
Results
Newly generated neurons in the hippocamal dentate gyrus showed a progressive reduction and became stable after 4 weeks. TUNEL-positive cells were detected in the subgranular zone (SGZ) and inner layer of the granule layer (GCL). The expression of human-bcl-2 gene can be detected both in immature and mature neurons. Bcl-2 Tg showed a significant increase in the number of BrdU-positive cells in the SGZ and GCL 30 days after BrdU injections compared with the littermates, both in the normal and ischemia. No difference in proliferation and differentiation were observed. Hippocampal culture also showed the enhanced survival of nascent BrdU-positive neurons in Bcl-2 Tg at 30 days after seeding.
Conclusion
Bcl-2 promotes newborn neurons survival in the hippocampal dentate gyrus under normal and ischemic conditions. These results indicate that modulation of Bcl-2 levels in combination with neurotrophins may have implications for therapeutic intervention to enhance neurogenesis for functional restoration, particularly after ischemia.