Arf4 Regulates Dendritic Spine Development Figure 9. Knockdown of Arf4 by shRNA reduces spine density in mouse primary neurons. (A) To verify the efficacy of the Arf4 shRNA- encoding plasmid, mouse Neuro-2A cells were transfected with Arf4-shRNA1 or Arf4-shRNA2 and equal protein amounts of transfected cell lysates were analyzed by immunoblotting with Arf4 antibody, or with actin antibody as a control. (B) Quantification of knockdown efficacy by Arf4-shRNA1 or Arf4-shRNA2 in mouse Neuro-2A cells. Arf4 levels are normalized to actin. (C–F9) Representative examples of WT neurons transfected with FUGW2- GFP plasmid (C–C9), Arf4-shRNA1 (D–D9), Arf4-shRNA2 (E–E9), or Arf4-shRNA plus Arf4-HA (rescue) (F–F9). Lower (C–F) and higher (C9–F9) magnification images are shown (N = 7–13 neurons per condition). (G) Total spine and (H) spine subtype densities from each of the experimental conditions. All data are mean6SEM. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0046340.g009 either human Arf4-HA and GFP-b-actin or GFP-b-actin alone. Overexpression of GFP-b-actin does not impair neuronal function or dendritic spine morphology and density and can, therefore, be used to highlight dendritic spines for imaging and quantification [29,45]. Overexpression of Arf4 did not significantly alter total dendrite number or length, nor did it change the number or length of primary, secondary, or tertiary dendrites (Figs. 6J–M). However, Arf4 overexpression dramatically increased dendritic spine density at DIV12 (Figs. 7B and B9), DIV14 (Figs. 7D and D9), and DIV19 (Figs. 7F and F9) compared with control neurons at each time point (Figs. 7A, C, and E). Furthermore, this increase in spine density was significant across time points, whereas spine densities remained relatively constant throughout development for neurons transfected with GFP-b-actin alone (Fig. 7G). We observed similar increases in spine density in GFP-b-actin co- transfection experiments using Arf4-mCherry in the place of Arf4- HA. The physiological effects of small GTPases depend on whether they are in a functionally active (GTP-bound) or inactive (GDP- bound) state [30]. To address the effect of Arf4 activity on spine density and morphology, we expressed a series of Arf4 functional mutants in cultured neurons. The Arf4 mutants used included the constitutively active mutant Arf4-Q71L, which remains bound to GTP, and the inactive mutant Arf4-T31N, which remains GDP- bound [36,46]. Arf4-Q71L had an even more pronounced effect on stimulating spine development than wildtype Arf4, whereas Arf4-T31N did not enhance spine development compared with controls (Figs. 8A and B). Arf4 and its active mutant also promoted stubby and thin spine development (Fig. 8C). Overexpression of the inactive Arf4-T31N mutant significantly reduced mushroom PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e46340