a group of hematological and solid tumors (reviewed by [18]). Due to the fact that many cancer cells manifest a stem cell- or progenitor-like gene expression pattern [19], it was not surprising that ABC transporter expression has been detected in a variety of organ-specific stem cells throughout the organism (reviewed in [20]). Hematopoietic stem cells show a reduced staining profile for the mitochondrial marker Rhodamine 123, which is a substrate of ABCB1, indicating high expression levels of ABCB1 in these stem cells [21]. Using fluorescence-activated cell sorters and bone marrow cells stained with fluorescent dyes such as Rhodamine 123 and Hoechst 33342, a small, low-profile subset of cells with phenotypic markers of hematopoietic stem cells was revealed [22]. This population is lost in the Hoechst profile after administration of the ABCB1 inhibitor verapamil [22,23], and was later confirmed in non-hematopoietic cell populations such as pancre- atic mesenchymal stem cells [24]. Mice lacking ABCB1 or ABCG2 do not show altered phenotypes [25,26] except for higher brain concentrations of ABCB1-transported agents/ligands, an in- creased immunological response to normal bacterial flora (ABCB10/0) [27], and a distinct form of dietary-induced proto- porphyria (ABCG20/0) [28]; however, these studies did not investigate neurogenic or cognitive functions. Recently, in vitro studies showed high expression rates of ABCB1 [29] and ABCG2 [30] in neurospheres during proliferation. The expression of both ABC transporters decreased substantially with the advancing state of differentiation, again suggesting a functional involvement of ABC transporters in stem cell homeostasis [31]. Considering the increasing number of observations indicating a functional role of ABC transporters in stem cell maintenance and differentiation, mostly from in vitro studies, we hypothesized that neurogenic processes would be impaired due to the deficiency of distinct ABC transporters. We focused on the transporters ABCB1 (MDR1, subfamily B) and ABCG2 (BCRP, subfamily G) based on the studies of Islam and colleagues [29,30], who first observed the transient expression of ABC transporters in NSPCs. Furthermore, we chose ABCC1- (MRP1, subfamily C) deficient mice because little information is available about its function in neurogenic processes, and because we recently showed its potential signifi- cance for brain homeostasis in proteopathies such as Alzheimer’s disease [17]. In the present study, we focused on the assessment of possible impairments of neurogenesis and cognitive alterations in vivo and cell-intrinsic effects on NSPC functions in vitro with regard to deficiency for the distinct ABC transporters. After comparative analyses of ABC transporter-deficient mouse strains and tissue in vivo and in vitro, we can confirm that a deficiency of ABC transporters leads to distinct impairments in NSPC maturation and adult neurogenesis in vivo. However, the in vitro experiments to determine proliferation and differentiation changes of NSPCs revealed that the underlying processes are not related to single stem cells. Consistent with the neurogenic alterations, we also show that ABC transporter-deficiency leads to far-ranging functional changes, even resulting in distinct behavioral phenotypes in a set of basic behavioral tests. Results ABC transporter-deficiency affects neuronal differentiation of NSPCs To assess the influence of ABC transporter-deficiency on neurogenesis, we analyzed molecular markers of neurogenic progenitors or immature neurons in the subgranular zone (SGZ) of the dentate gyrus (DG) in adult mice aged 3–4 months. We characterized marker proteins to quantify the differentiation states of NSPCs. To label very early stem and progenitor cells, we used ABC Transporters in Neurogenesis an antibody against Sox2, a transcription factor that is required for maintenance of stem cell status, and therefore a reliable marker of early undifferentiated NSPCs [32,33,34]. To quantify the number of early neuronal progenitors, we used the marker protein doublecortin (DCX) [35,36]. Postmitotic, newly generated neu- rons at a stage of axonal and dendritic targeting were labeled with an anti-calretinin antibody [37]. The quantification of the cells revealed a significant increase in Sox2+ early NSPCs only in ABCC10/0 (+17%) mice, whereas their number was not altered in ABCB10/0 or in ABCG20/0 mice as compared to the genetic background controls (FVB/N) (Figure 1A). Investigating early neuronal progenitors, which are positive for DCX, revealed a significant decrease of these cells in mice lacking ABCB1 (236%) or ABCG2 (223%), but no changes in ABCC10/0 mice (Figure 1B). Quantifying the number of post-mitotic, newly generated calretinin+ neurons, we also detected decreased cell numbers in ABCB10/0 animals (237%), while deficiency in ABCG2 or ABCC1 did not induce significant effects (Figure 1C). The proliferation capacity (BrdU+ cells) was not affected in any of the analyzed ABC transporter-deficient mice (Figure 1D). Thus, ABCC10/0 mice only revealed slightly increased numbers of early NSPCs, while the fate of differentiation was not affected. ABC transporter-deficiency alters the rate of NSPC differentiation To analyze Sox2, DCX and calretinin expression in the actively proliferating NSPC population, we double-labeled brain sections for BrdU and the respective marker protein. The mice received BrdU for 7 days prior to sacrifice. The analyses revealed decreasing amounts of newly proliferating Sox2+ NSPCs in the absence of each of the three ABC transporters (225% ABCB10/0; 228% ABCG20/0; 237% ABCC10/0; Figure 2A, 2D). The number of DCX+ neuronal progenitors did not show any ABC transporter-related changes in the proliferating population (Figure 2B, 2E). Interestingly, the population of post-mitotic, newly differentiated granule cells expressing calretinin significantly increased both in ABCG20/0 (+43%) and ABCC10/0 (+51%) mice, while the increase in ABCB10/0 mice did not reach statistical significance (Figure 2C, 2F). ABC transporter deficiency does not impair neurogenic functions in vitro To analyze the proliferation capacity of ABC transporter- deficient NSPCs, we used primary NSPC lines from the subventricular zone of control and ABC transporter-deficient animals. The proliferation capacity of NSPCs was measured by BrdU-uptake into the DNA of proliferating cells, and compara- tively quantifying the BrdU+ cell fraction of each cell line. The BrdU intake over a period of 30 min revealed a significant increase in proliferation activity in cell lines deficient in the ABC transporter ABCG2 (+62%) (Figure 3A, 3B). The increase of proliferation activity in ABCB10/0 and ABCC10/0 cells did not reach significance. To assess the differentiation potential of these primary NSPC lines, we let the cells differentiate for 3 days and quantified the number of differentiated neurons using immunofluorescence techniques. Comparing the fraction of Tuj1+, newly differentiated neurons among the different NSPC lines, we could not detect significant alterations (Figure 3C, 3D). Therefore we conclude that deficiency of ABCB1, ABCG2 or ABCC1 does not affect the ability of NSPCs to differentiate into neurons per se. PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35613