Arf4 Regulates Dendritic Spine Development Figure 1. Arf4+/2 mice are impaired in a dentate-gyrus dependent pattern separation task. All mice were between 4–5 months of age at the time of experimentation. (A) A map of the Arf4 gene trapping construct obtained from BayGenomics (ES cell line CSH658). Ex, exon; b-gal, b- galactosidase gene; neo, neomycin-resistance gene. (B) Hippocampi from three WT and Arf4+/2 littermate pairs were homogenized, followed by immunoblotting with anti-Arf4 (left panel). Actin loading controls are shown. Quantification of Arf4 protein levels in hippocampi prepared from WT or Arf4+/2 mice (right panel). Arf4 protein levels were normalized to actin. (C) X-gal stained sagittal brain section from an Arf4+/2 mouse at 5X magnification. (D) Representative image from a 4.5-month-old Arf4+/2 hippocampus. (E) Quantification of the amount of time male (left panel) or female (right panel) WT and Arf4+/2 mice spent exploring a novel object/context during a pattern separation task. (N = 12–13 mice per genotype per sex). (F–G) Quantification of the amount of time male (left panel) or female (right panel) WT and Arf4+/2 mice spent exploring an object in a specific context during the first (F) or second (G) trial of the pattern separation task. All data are mean6SEM. * p,0.05, ** p,0.01. doi:10.1371/journal.pone.0046340.g001 Here, we report that Arf4+/2 mice display impairments in a DG-dependent pattern separation task, as well as significant spine loss and smaller mEPSCs in their DG granule cells. Consistent with our in vivo findings, knocking down Arf4 decreases spine density in primary neurons, whereas Arf4 overexpression signif- icantly increases spine density. These effects are regulated by ASAP1, a GAP that was previously shown to form a complex with Arf4 and regulate GTP hydrolysis for Arf1, Arf4, and Arf5 [32,33]. Furthermore, Arf4 overexpression restores spine loss in an AD-related apoE4 transgenic mouse model, suggesting a potential therapeutic use for Arf4. Materials and Methods Generation of Arf4+/2 mice The Arf4+/2 mouse model was established based on an embryonic stem cell line from BayGenomics (CSH658). The ES cell line contains a gene trapping construct (pGT1lxf) in intron 1 of the Arf4 gene, located upstream of the gene encoding the b- galactosidase/neomycin-resistance fusion protein. The FastStart Taq DNA Polymerase dNTPack kit (Roche) was used to generate candidate forward primers designed for 200–500 base pair intervals of intron 1 of the Arf4 gene. One common reverse primer in the b- galactosidase reporter, RT416, was applied in all reactions (59- GTCCTCTGGTGCTCAAAGACC-39). Amplification with for- ward primer P8 (59-TGGAAGCACAGGCCTTTAATCC-39), located in intron 1, yielded a distinct PCR product of approximately 800 kb. PCR conditions were 34 cycles at 95uC for 30 s, 57uC for 30 s, and 72uC for 1 min, followed by a final extension at 72uC for 7 min. The PCR product was verified by sequencing. The CSH658 ES cells were microinjected into C57Bl6 blastocysts to yield chimeras, which were identified by coat color. A chimeric male was crossed with wildtype C57Bl6 females, and germline transmission resulted in heterozygote males and females PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e46340