ASAP1 might interact to influence spine cytoskeletal dynamics. Recent studies have shown that Arf activation promotes recruit- ment of several actin-regulatory proteins, including cortactin and dynamin, to the vesicle-budding sites of the trans-Golgi network (TGN) [62,63]. Our finding that Arf4 colocalizes with actin in primary neurons suggests a potential interplay between Arf4 and actin-regulating proteins in dendritic spines. Role of Arf4 in dentate gyrus-dependent pattern separation tasks Mossy fiber ‘‘detonator’’ synapses arising from DG granule cells strongly activate the CA3 region and are involved in a variety of neurological functions, including memory and spatial representa- tions [6,64]. Our experiments showed that Arf4 heterozygosity in vivo leads to reductions in spine number and mEPSC amplitude in the DG, and these alterations accompany profound pattern separation impairments. These results indicate that Arf4 could serve as a novel regulator of the mossy fiber pathway by regulating granule cell spine development and electrophysiological activity. Spine head size is positively correlated with mEPSC amplitude [65], and the functional loss of NMDA receptors in DG granule cells has been associated with impairments in pattern separation [8]. In our study, mushroom spine numbers were significantly lower in the DG of Arf4+/2 compared to WT mice, and this spine loss correlated with a reduction in mEPSC amplitude. Mushroom spines contain more AMPA and NMDA receptors than other spine types, and these receptors are critical for strengthening synaptic connections [14]. Therefore, the context recognition impairments seen in Arf4+/2 mice could be related to reduced granule cell-specific synaptic communication caused by the loss of mushroom-type spines. Arf4 overexpression as a potential therapeutic strategy for AD-related spine loss As spine loss is strongly correlated with cognitive impairments in AD [15,66], a potential therapeutic strategy for restoring cognitive References 1. Deng W, Aimone JB, Gage FH (2010) New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11:339–350. 2. Squire LR, Stark CE, Clark RE (2004) The medial temporal lobe. Annu Rev Neurosci 27:279–306. 3. Schmidt B, Marrone DF, Markus EJ (2011) Disambiguating the similar: the dentate gyrus and patern separation. Behav Brain Res 226:56–65. 4. Aimone JB, Deng W, Gage FH (2011) Resolving new memories: a critical look at the dentate gyrus, adult neurogenesis, and pattern separation. Neuron 70:589– 596. 5. Treves A, Tashiro A, Witter ME, Moser EI (2008) What is the mammalian dentate gyrus good for? Neuroscience 154:1155–1172. 6. Yassa MA (2011) Pattern separation in the hippocampus. Trends Neurosci 34:515–525. 7. Gilbert PE, Kesner RP, Lee I (2001) Dissociating hippocampal subregions: double dissociation between dentate gyrus and CA1. Hippocampus 11:626–636. 8. McHugh TJ, Jones MW, Quinn JJ, Balthasar N, Coppari R, et al. (2007) Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317:94–99. 9. Yuste R (2011) Dendritic spines and distributed circuits. Neuron 71:772–780. 10. Hotulainen P, Hoogenraad CC (2010) Actin in dendritc spines: connecting dynamics to function. J Cell Biol 189:619–627. 11. Hering H, Sheng M (2001) Dendritic spines: structure, dynamics, and regulation. Nat Rev Neurosci 2:880–888. 12. Segal M (2005) Dendritic spines and long-term plasticity. Nat Rev Neurosci 6:277–284. 13. Peebles CL, Yoo J, Thwin MT, Palop JJ, Noebels JL, et al. (2010) Arc regulates spine morphology and maintains network stability in vivo. Proc Natl Acad Sci USA 107:18173–18178. 14. Bourne J, Harris KM (2007) Do thin spines learn to be mushroom spines that remember? Curr Opin Neurobiol 17:381–386. 15. Penzes P, Cahil ME, Jones KA, VanLeeuwen JE, Wolfrey KM (2011) Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 14:285–292. Arf4 Regulates Dendritic Spine Development function in AD patients could be to increase dendritic spine density, thereby strengthening synaptic connections. In our current study, we found that overexpression of Arf4 restored spine loss in NSE-apoE4 neurons. Thus, increasing Arf49s function might serve as a potential therapeutic strategy for restoring impairments in spine development and synaptic connectivity. The preclinical period prior to the diagnosis of AD is characterized by deficits in a number of memory-related processes, including pattern separation [64,67]. Patients with amnestic mild cognitive impairment (MCI), for instance, showed an impaired ability to distinguish between a previously seen object and a very similar but unobserved object [68]. Some aged rodents that do not develop AD pathology nevertheless have memory deficits, such as a failure to encode novel information while navigating similar situations [69]. In our current study, it is intriguing that young (4– 5 month old) Arf4+/2 mice have pattern separation deficits that are similar to those found in pre-clinical MCI patients and aged rodents, but exhibit normal spatial learning and memory. The Arf4+/2 mouse model might therefore represent a unique tool to investigate early cognitive dysfunctions prior to the onset of AD pathology. Our studies should provide further understanding of the molecular mechanisms underlying DG-dependent memory tasks and spine development, and how these processes are degraded in neurodegenerative disorders, such as AD. Acknowledgments We thank Drs. Steven Finkbeiner and Eric Huang for critical discussions. We also thank Dr. Nino Devidze, Iris Lo, and Aubrey Bernardo for technical assistance, Gary Howard and Dr. Anna Lisa Lucido for editorial assistance, and Linda Turney for manuscript preparation. Author Contributions Conceived and designed the experiments: SJ YH. Performed the experiments: SJ SYY LZ JB JD DW YH. Analyzed the data: SJ LZ JD DW YH. Wrote the paper: SJ LZ YH. 16. Fiala JC, Spacek J, Harris KM (2002) Dendritic spine pathology: cause or consequence of neurological disorders? Brain Res Rev 29:29–54. 17. Tackenberg C, Ghori A, Brandt R (2009) Thin, stubby or mushroom: spine pathology in Alzheimer’s disease. Curr Alzheimer Res 6:261–268. 18. Dumanis SB, Tesoriero JA, Babus LW, Nguyen MT, Trotter JH, et al. (2009) ApoE4 decreases spine density and dendritic complexity in cortical neurons in vivo. J Neurosci 29:15317–15322. 19. Ji Y, Gong Y, Gan W, Beach T, Holtzman DM, et al. (2003) Apolipoprotein E isoform-specific regulation of dendritic spine morphology in apolipoprotein E transgenic mice and Alzheimer’s disease patients. Neuroscience 122:305–315. 20. Brodbeck J, Balestra ME, Saunders AM, Roses AD, Mahley RW, et al. (2008) Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons. Proc Natl Acad Sci USA 105:1343–1346. 21. Brodbeck J, McGuire J, Liu Z, Meyer-Franke A, Balestra ME, et al. (2011) Structure-dependent impairments of intracellular apolipoprotein E4 trafficking and its detrimental effects are rescued by small-molecule structure correctors. J Biol Chem 286:17217–17226. 22. Andrews-Zwilling Y, Bien-Ly N, Xu Q, Li G, Bernardo A, et al. (2010) Apolipoprotein E4 causes age-and tau-dependent impairment of GABAergic interneurons, leading to learning and memory deficits in mice. J Neurosci 30: 13707–13717. 23. Tada T, Sheng M (2006) Molecular mechanisms of dendritic spine morphogenesis. Curr Opin Neurobiol 16:95–101. 24. Lippman J, Dunaevsky A (2005) Dendritic spine morphogenesis and plasticity. J Neurobiol 64: 47–57. 25. Tolias KF, Duman JG, Um K (2011) Control of synapse development and plasticity by Rho GTPase regulatory proteins. Prog Neurobiol 94:133–148. 26. Klassen MP, Wu YE, Maeder CI, Nakae I, Cueva JG, et al. (2010) An Arf-like small G protein, ARL-8, promotes the axonal transport of presynaptic cargoes by suppressing vesicle aggregation. Neuron 66:710–723. 27. Choi S, Ko J, Lee JR, Lee HW, Kim K, et al. (2006) ARF6 and EFA6A regulate the development and maintenance of dendritic spines. J Neurosci 26:4811–4819. PLOS ONE | www.plosone.org 14 September 2012 | Volume 7 | Issue 9 | e46340