It is well known that the development of the human brain is accompanied by a massive loss of neuronal cells by apoptotic processes and apoptosis has been widely observed in incompletely differentiated neuronal cells in vivo. The human neuronal progenitor cell line ReNCell VM197 can be easily driven into differentiation by removal of growth factors (EGF, bFGF) and it differentiates within few days into oligodendrocytes, astrocytes and neurons and is therefore a suitable model to investigate apoptotic processes during differentiation of human neuronal cells.

We studied the protein status and the expression level of caspases and Bcl-2 family members during differentiation of VM197 cells in vitro. UV-B and staurosporine-exposed cells served as positive controls.

A significant decrease in cell number was observed during the first 12 h of differentiation. The levels of caspase-7 and caspase-2 were diminished throughout differentiation as determined by immunoblotting. However, activated caspase-3 (p17, p11) was detected 4 h up to 72 h post-initiation of differentiation and correlated with increasing numbers of apoptotic cells as measured by flow cytometry (AnnexinV-FITC/PI). In contrast, at later stages of differentiation CAS (cellular apoptosis susceptibility gene) protein expression was strongly diminished and the expression of Bax was decreased and a cleaved form of Bax was observed. Additionally, the expression level of Bcl-2 was found to be increased.

We conclude that apoptosis occurs during differentiation of VM197 cells and that in fully differentiated VM197 cells Bcl-2 plays a protective role against apoptosis. Furthermore apoptosis susceptibility is reduced by a decreased expression of pro-apoptotic proteins.