These speculations prompted us to examine the level of BAG3 expression in adult rat hippocampal neural progenitors. As shown in Figure 1A (lane 1), passage 25 neural progenitor cells, isolated from hippocampus and characterized elsewhere, 1 expressed BAG3 protein at moderate levels when cultured in Neurobasal medium supplemented with N2 plus FGF2. When cells were subjected to FGF2-starvation, BAG3 expression dropped by 60%(lane 2). The addition of FGF2 to culture media devoid of the growth factor restored the basal level of BAG3 (lane 3), suggesting that FGF2 sustains BAG3 expression in neural progenitor cells. This data is in accordance with earlier observations on SK-N-MC neuroblastoma cells. 7 The overnight FGF2 starvation, though, did not change the neural progenitor features of the cells, as verified by qPCR analysis of the markers of stemness (SOX2 and Nestin) and neural lineages (type III β-tubulin, GFAP, O4)(data not shown). As FGF2 plays an important role in the proliferation of neural progenitors, we sought to investigate the impact of FGF2-driven BAG3 expression on the growth rate of neural stem cells. To this end cells were treated with an adenovirus expressing siRNAs targeting the BAG3 mRNA and three days after infection protein analysis and cell number were measured. As shown in Figure 1B, treatment of neural progenitor cells with Ad-siBAG3, but not Ad-null, suppressed BAG3 expression in the presence of FGF2. Accordingly, reduced level of BAG3 determined a decrease in the cell number greater than 45%, indicating that BAG3 does have a role in the proliferation rate of neural progenitor cells.

In the next series of experiments we investigated the potential of the mitogens FGF2 and EGF to stimulate BAG3 expression in secondary neurospheres extracted from the developing brain of mouse embryo. As described previuosly, 11 primary neurospheres are non-adherent spherical structrures mainly constituted of nestin-positive cells (neural stem cells) which maintain their primitive ontogenetic state when cultured in presence of mitogens such as FGF2 and EGF, and in the absence of substrate attachment. Secondary neurospheres were obtained as described in the Figure legend. After a 10 h starvation from growth factors, the cells were readministrated with FGF2 or EGF or FGF2/EGF added together. As shown in Figure 1C, the level of 80 KDa BAG3 protein was undetectable when secondary neurospheres were starved from mitogens (lane 1). 48 h after readministration, BAG3 expression was noticeably induced at comparable levels either in FGF2-(lane 2), EGF-(lane 3) or EGF/FGF2-treated cells (lane 4). To confirm that the stimulatory effect of FGF2 on BAG3 expression occurred in nestin-positive cells, secondary neurospheres starved of growth factors (left part) or readministrated with FGF2 (right part) were double labeled with BAG3 antibody and the progenitor marker nestin antibody (Fig. 1D). According to western blot data, mitogen-starved neural progenitors display undetectable BAG3 levels in nestin positive cells, while Rhodamine-labeled BAG3 antibody colacalizes with FITC-labeled nestin antibody in FGF2-treated cells. These observations confirm that BAG3 induction upon FGF2 treatment