Arf4 Regulates Dendritic Spine Development Figure 12. Arf4 overexpression rescues spine loss in apoE4-expressing primary neurons. (A) Representative Western blot of actin and Arf4 levels in NSE-apoE4 and -apoE3 mice reveal a decrease in Arf4 protein levels from NSE-apoE4 mice compared to those from NSE-apoE3 mice. Primary neuron lysates were prepared in triplicate, and actin was used as a loading control. (B) Quantification of the ratio of Arf4/Actin levels. Data are mean6SD. * p,0.05, *** p,0.001. (C–H9) Primary hippocampal and cortical neurons were cultured from WT, homozygous NSE-apoE3, or homozygous NSE-apoE4 mouse E182E19 embryos. Neurons were transfected with FUGW2 plasmid alone (for visualization of spines) or with FUGW2 plus Arf4-HA-WT at DIV5 and analyzed at DIV14. Lower (C2H) and higher (C92H9) magnification images are shown. (C–E9) WT (C–C9), NSE-apoE3 (D– D9), or NSE-apoE4 (E–E9) neurons transfected with empty FUGW2 plasmid (n = 12–13 neurons). (F–H9) WT (F–F9), NSE-apoE3 (G–G9), or NSE-apoE4 (H– H9) neurons cotransfected with FUGW2 plus Arf4-HA-WT (n = 8–12 neurons). (I) Quantification of total spine densities for each experimental condition. (J) Spine subtype densities for each experimental condition. All data are mean6SEM. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0046340.g012 stabilize after LTP [52,53]. Mushroom spines have larger postsynaptic densities (PSDs) and more glutamate receptors than thin spines, making the synapse functionally stronger and more stable [14,23]. We found that knocking down Arf4 decreases thin and mushroom spine density and, consistently, impairs the electrophysiological function of granule cells of the dentate gyrus. Decreases in both spine types have been reported in animal models of cognitive decline [54,55]. Based on these results, a significant loss of both transient thin spines and stable mushroom spines in primary neurons lacking Arf4 raises the possibility that Arf4 might be critical for both dendritic spine plasticity and stability. Our study uncovered potential molecular mechanisms govern- ing the effects of Arf4 on spine density and morphology. First, we found that the effects of Arf4 on spine development are related to its activity state. The GTP-bound form of Arf4 stably associates with intracellular organelle membranes and can activate down- stream effectors [56,46]. By constitutively binding to GTP, Arf4- Q71L likely activates signaling pathways that can induce spine morphogenesis to a greater extent than wildtype Arf4. In contrast, Arf4-T31N remains bound to GDP and is therefore theoretically inactive. Previous studies have reported seemingly contradictory roles for GDP-bound Arf proteins (Arf-T31N) as either inactive mutants whose physiological effects do not differ from wildtype Arfs [57,58], or as dominant-negative mutants with inhibitory effects on vesicular transport and other cellular functions [59]. Interestingly, our results suggest that Arf4-T31N acts as an inactive mutant with respect to its effects on total spine density, but displays some dominant-negative characteristics by specifically reducing the density of mushroom spines. Small GTPases modulate actin cytoskeletal rearrangement and dynamics in both neuronal and non-neuronal cells [10,60], suggesting that actin-binding proteins or their regulators might serve as effectors of Arf4 in spine morphogenesis. We found that the Arf GAP ASAP1 negatively regulates dendritic spine density compared to controls, and this effect is partially rescued by co- expression of ASAP1 together with Arf4-WT. ASAP1 is known to regulate the actin cytoskeleton [61], indicating that Arf4 and PLOS ONE | www.plosone.org 13 September 2012 | Volume 7 | Issue 9 | e46340