ABC Transporters in Neurogenesis Figure 7. ABC transporter-deficiency influences cortical architecture of layers I and VI while neuronal density is not altered. Paraffin- embedded brain sections (A) were labeled with an antibody against NeuN, digitized, and the NeuN+ area was computer-assisted determined relative to the cortical area to quantify the neuronal density of the cortex (B). The thickness of the cortical layers I, II–IV (combined), V and VI were measured (A) and analyzed (C). The loss of ABCB1 expression significantly decreased the thickness of layer I (212%) while loss of ABCC1 only affected the thickness of layer VI (+15%) Scale bars: 500 mm (A); Error bars: SEM, *p,0.05; **p,0.01. doi:10.1371/journal.pone.0035613.g007 are of broad interest in that they may be linked to neurodegen- erative diseases, stroke or the effects of brain aging. With regard to aging, it is important to consider the physiological function of ABC transporters not only at barriers and epithelia, but also in the brain’s NSPC pool. The ABC transporters convey a wide spectrum of biological and metabolic substrates [2,3,4]. The impairment of ABC-mediated excretion across the BBB might alter the microenvironment of the stem cell niche, which is located directly between the vascular and the ventricular system. The interaction of the NSPCs with both systems thereby implies changes in the NSPC’s homeostasis due to altered ABC transport [49]. In the absence of sufficient ABC transport, metabolites and chemical compounds can reach functional and/or pathogenic concentrations within the cells, and thus influence the NSPC pool or its differentiation behavior. For instance, it has been shown that endothelial cells are able to generate soluble factors that can influence the differentiation of NSPCs in vitro [50]. Assuming that some of these factors are substrates for ABC transporters, their diminished elimination by transporters could adversely influence both the microenvironment of the stem cell niche and cellular homeostasis, thereby altering NSPC functions. Our investigations of the proliferation capacity and ability to differentiate into neuronal lineage in vitro did not support the idea of a cell-intrinsic function of any of these ABC transporters, directly influencing the cell fate [29,30,31]. While we found that NSPCs appear to benefit from the absence of ABCG2 expression, as evidenced by higher cell proliferation, our differentiation experiments did not reveal any impairment due to ABC transporter-deficiency. In particular, the increased proliferation capacity of NSPCs lacking ABCG2 expression does not easily integrate into the observations made by analyzing neurogenic functions in the DG. Rather, this experiment supports the idea of ABC transporter-deficiency induced changes in the global microenvironment of the brain. Furthermore, this altered microenvironment could lead to the observed functional changes in neurogenesis and even behavior. While our mRNA expression analyses confirmed that all ABC transporters investigated are expressed in NSPCs (except the murine ABCB1 subtype ‘‘a’’), we also found evidence of possible co-regulation of the expression levels of specific ABC transporters. While the lack of ABCB1b expression augmented both ABCG2 and ABCC1 expression, in contrast, the loss of ABCG2 only had a small lowering effect on ABCB1b expression, and deficiency of PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35613