ABC Transporters in Neurogenesis Figure 1. Deficiency of ABCB1 impairs neuronal differentiation in the SGZ of the dentate gyrus. Shown are the relative quantities of cells positive for Sox2, DCX, calretinin and BrdU in the SGZ of the dentate gyrus in ABC transporter-deficient mice as compared to the FVB controls. A) Sox2+ NSPCs increased significantly in ABCC1 transporter-deficient mice (+17%), while ABCB1- or ABCG2-deficient mice were unaffected. B) Early DCX+ neurons were significantly decreased in the absence of ABCB1 (236%) and ABCG2 (223%), while later, post-mitotic calretinin+ cells (C) were only reduced in ABCB1 transporter-deficient mice by 37%. D) The proliferation capacity (BrdU+ cells) was not affected in any of the analyzed ABC transporter-deficient mice. Error bar: SEM; * p,0.05; **p,0.01. doi:10.1371/journal.pone.0035613.g001 ABC transporter expression in NSPCs To confirm whether ABC transporters are expressed in pro- liferating NSPCs, we used TaqManH assays to quantify relative expression levels for ABCB1a, ABCB1b, ABCG2 and ABCC1 in background controls and ABC transporter knockout strains. Quantitative RT-PCR analyses with NSPC mRNA confirmed that all three transporters (B1, C1, G2) are expressed, and also indicated a possible co-regulation between ABCB1 and ABCG2. We found only one form (ABCB1b) of the murine ABCB1 transporter expressed in NSPCs, while ABCB1a mRNA was not detectable. Analyses showed that the lack of ABCG2 led also to a significant decrease of ABCB1 mRNA amounts (228%) (Figure 4A). In contrast, loss of ABCB1b expression led to a marked increase in ABCG2 mRNA (+71%) (Figure 4B). We also found ABCG2 mRNA levels increased by 73% as a result of ABCC1 deficiency, although this increase did not reach statistical significance (Figure 4B). On the other hand, ABCC1 deficiency did not lead to significant effects on the expression of ABCB1 or ABCG2. However, ABCC1 expression itself seemed not to be altered by the loss of neither ABCB1 nor ABCG2 expression (Figure 4C). ABC transporter-deficiency reduces neurogenesis in disease states Traumatic brain injury is known to activate the proliferation and differentiation of resident NSPCs [38]. Since ABC trans- porter-deficiency under normal physiologic conditions causes only minor phenotypic changes, we wanted to assess the potential importance of the transporters during disease states. The controlled cortical impact (CCI) paradigm [39] was chosen to amplify minor effects of ABC transporter involvement in NSPC homeostasis with regard to proliferation and differentiation capacity. Therefore, cortical trauma was induced 7 days prior to analysis. In agreement with recent publications [38,39,40], neurogenic processes were significantly increased in the dentate gyrus of the induced (injured) hemisphere compared to the contralateral control hemisphere in all analyzed strains (Figure 5). CCI-induced trauma resulted in a highly increased cell proliferation in the SGZ of the affected hemisphere in all analyzed mouse strains. The proliferation index of the ipsilateral hemi- spheres was significantly increased as compared to the contralat- eral, untreated control hemispheres in all mice. Importantly, ABC transporter deficiency substantially altered the extent of the proliferation increase. Relative to the control group, we observed significantly lower ipsilateral proliferation indices in all three ABC transporter-deficient strains (247% ABCB10/0; 231% ABCG20/ 0; 253% ABCC10/0, Figure 6A). Consistent with the highly increased cell proliferation, we also detected an expansion in the analyzed subpopulations after CCI-treatment. CCI led to in- creased numbers of Sox2+ NSPCs in the ipsilateral hemisphere independent of ABC transporter expression (Figure 6B). However, the CCI paradigm mostly affects the number of differentiating cells of the neuronal lineage that are DCX+ and calretinin+. All mice showed significantly more differentiating DCX+ neuronal pro- genitors in the induced hemisphere as compared to the control hemispheres. Comparing controls and ABC transporter-deficient strains, only ABCB10/0 and ABCC10/0 mice exhibited a sub- stantial decrease in the number of differentiating DCX+ neuronal progenitors (236% ABCB10/0; 233% ABCC10/0), while the cell number was not affected in ABCG20/0 mice (Figure 6C). Analogous to the situation of the DCX+ cell pool, we also detected significantly lower numbers of calretinin+ postmitotic PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35613