Arf4 Regulates Dendritic Spine Development Figure 11. Arf4 levels do not affect BDNF expression in vivo or in vitro. (A2B) Representative images of WT (A) and Arf4+/2 (B) hippocampi stained for BDNF, followed by DAB development. (C) Quantification of BDNF immunoreactivity in the hippocampus (n = 3 mice per genotype). (D2E) Duplicate images of primary neurons transfected with Arf4-HA at DIV5 and stained with HA (green), BDNF (red), and MAP2 (blue) antibodies at DIV14. Arrows point to a neuron overexpressing Arf4-HA (white arrow) or a neuron that does not overexpress Arf4-HA (pink arrow) (F) Quantification of BDNF expression in primary neurons either overexpressing or not overexpressing human Arf4-HA (n = 15 neurons per condition). A.U. = arbitrary units. doi:10.1371/journal.pone.0046340.g011 changes in spine density and morphology, suggesting that ASAP1 is a negative regulator of Arf49s effects on spine density. BDNF levels are not altered in Arf4+/2 mice or in Arf4- overexpressing primary neurons Since brain-derived neurotrophic factor (BDNF) plays critical roles in both pattern separation [47] and dendritic spine development [48,49], we asked whether BDNF expression might be altered in Arf4+/2 mice. We found that BDNF protein levels in the CA1, CA3, and DG regions of the hippocampus were similar in Arf4+/2 mice compared to their wildtype littermates (Fig. 11A–C). BDNF levels also remained unaltered in Arf4-overexpressing neurons compared to neurons not overexpressing Arf4 (Fig. 11D–F). Thus, Arf4 levels do not appear to have a direct effect on neuronal BDNF expression in vitro or in vivo. Arf4 overexpression rescues spine loss in neurons from an AD-related apoE4 mouse model We next examined Arf49s role in spine development in the context of a neurodegenerative disease model. We investigated the possibility that Arf4 might rescue apoE4-caused spine loss in neurons from transgenic mice expressing apoE4 selectively in neurons [neuron-specific enolase (NSE)-apoE4]. These mice have impairments in learning and memory [50], as well as a loss of dendritic spines in primary neurons and in the hippocampus and cortex [21]. We found that Arf4 mRNA levels were significantly reduced in hippocampi from 10-month-old female NSE-apoE4 (vs. NSE-apoE3) mice (36% reduction, p = 0.015). Furthermore, Arf4 protein levels were also significantly reduced in primary neurons from NSE-apoE4 mice compared to those from NSE- apoE3 mice (Figs. 12A and B). Consistent with previous findings [21], the dendritic spine density of NSE-apoE4 neurons (Figs. 12E and E9) was significantly less than that of either NSE-apoE3 (Figs. 12D and D9) or WT neurons (Figs. 12C and C9). Additionally, NSE-apoE4 neurons had fewer stubby, thin, and mushroom spines compared with WT neurons (Fig. 12J). Overexpression of Arf4 fully restored the spine loss observed in NSE-apoE4 neurons (Figs. 12H and H9). Furthermore, Arf4 overexpression increased spine density to an extent similar to that observed in Arf4-overexpressing WT (Figs. 12F and F9) and NSE-apoE3 neurons (Figs. 12G and G9). The apoE4-induced decrease in specific spine subtypes was also rescued by Arf4 overexpression (Fig. 12J). Discussion In this study, we show that the small GTPase Arf4 is a novel modulator of DG-dependent pattern separation tasks by regulating dendritic spine development. The loss of one copy of Arf4 in vivo leads to severe impairments in pattern separation, as well as a decrease in DG granule cell spine density and mEPSC amplitude. In primary neuron cultures, overexpression of wildtype Arf4 promotes spine development even at an early stage (DIV12), whereas shRNA knockdown of Arf4 inhibits it. These effects are partially mediated by ASAP1, an Arf4 GAP. In addition, overexpression of Arf4 rescues the dendritic spine loss caused by apoE4, the major genetic risk factor for AD. These results indicate that Arf4, by promoting spine development, represents a useful target for treatments of neurodegenerative diseases that cause profound synaptic loss, such as AD. Arf4 regulates dendritic spine density and morphology Spine number and structural plasticity are tightly correlated with synaptic function in the mammalian brain [23,51]. We found that Arf4 overexpression promotes spine development, particularly that of thin spines. Previous studies have shown that thin spines are more transient and motile than mushroom spines, and an increase in the proportion of thin spines represents a greater capacity to PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e46340