Arf4 Determines Dentate Gyrus-Mediated Pattern Separation by Regulating Dendritic Spine Development Sachi Jain1,3, Seo Yeon Yoon1, Lei Zhu1, Jens Brodbeck1, Jessica Dai1, David Walker1,2, Yadong Huang1,2,3,4,5* 1 Gladstone Institute of Neurological Disease, San Francisco, California, United States of America, 2 Gladstone Institute of Cardiovascular Disease, San Francisco, California, United States of America, 3 Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, California, United States of America, 4 Department of Neurology, University of California San Francisco, San Francisco, California, United States of America, 5 Department of Pathology, University of California San Francisco, San Francisco, California, United States of America Abstract The ability to distinguish between similar experiences is a critical feature of episodic memory and is primarily regulated by the dentate gyrus (DG) region of the hippocampus. However, the molecular mechanisms underlying such pattern separation tasks are poorly understood. We report a novel role for the small GTPase ADP ribosylation factor 4 (Arf4) in controlling pattern separation by regulating dendritic spine development. Arf4+/2 mice at 4–5 months of age display severe impairments in a pattern separation task, as well as significant dendritic spine loss and smaller miniature excitatory post- synaptic currents (mEPSCs) in granule cells of the DG. Arf4 knockdown also decreases spine density in primary neurons, whereas Arf4 overexpression promotes spine development. A constitutively active form of Arf4, Arf4-Q71L, promotes spine density to an even greater extent than wildtype Arf4, whereas the inactive Arf4-T31N mutant does not increase spine density relative to controls. Arf49s effects on spine development are regulated by ASAP1, a GTPase-activating protein that modulates Arf4 GTPase activity. ASAP1 overexpression decreases spine density, and this effect is partially rescued by concomitant overexpression of wildtype Arf4 or Arf4-Q71L. In addition, Arf4 overexpression rescues spine loss in primary neurons from an Alzheimer’s disease-related apolipoprotein (apo) E4 mouse model. Our findings suggest that Arf4 is a critical modulator of DG-mediated pattern separation by regulating dendritic spine development. Citation: Jain S, Yoon SY, Zhu L, Brodbeck J, Dai J, et al. (2012) Arf4 Determines Dentate Gyrus-Mediated Pattern Separation by Regulating Dendritic Spine Development. PLoS ONE 7(9): e46340. doi:10.1371/journal.pone.0046340 Editor: Zhongcong Xie, Massachusetts General Hospital, United States of America Received June 17, 2012; Accepted August 29, 2012; Published September 25, 2012 Copyright: 2012 Jain et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from the National Institute of Health (P01 AG022074, P30 NS065780, and C06RR18928), the National Science Foundation, the J. David Gladstone Institutes, and gifts from the S.D. Bechtel, Jr. Foundation and the Hellman Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yhuang@gladstone.ucsf.edu Introduction In the adult mammalian brain, the hippocampus plays a central role in the encoding and storage of certain types of memory, including spatial and episodic memory [1,2]. The dentate gyrus (DG) subregion of the hippocampus mediates episodic memory formation and the disambiguation of similar but discrete events, a phenomenon known as pattern separation [3,4,5,6]. Behavioral studies have shown that animals with impaired DG function are unable to differentiate between similar events or objects [7,8], providing empirical evidence for a role of the DG in differentiating memories. Like most excitatory neurons, granule cells of the DG are covered by dendritic spines that are important loci of synaptic plasticity [9,10,11,12]. Spine morphology correlates with synaptic strength and structural plasticity; for instance, thin spines are highly motile and likely to respond to activity-induced changes, whereas mushroom spines have larger post-synaptic densities (PSDs) and are more stable [13,14]. Aberrations in spine density and morphology are associated with a number of neurological disorders, including Alzheimer’s disease (AD) [15,16,17]. In particularly, apolipoprotein (apo) E4–a major genetic risk factor for Alzheimer’s disease–decreases spine density both in vivo [18,19] and in vitro [20,21], and is associated with spatial learning and memory impairments [22]. At the molecular level, small GTPases play a critical role in neuronal trafficking and spine morphogenesis [23,24,25,26]. Mem- bers of the ADP-ribosylation factor (ARF) family of small GTPases and their regulators have gained increasing attention as important regulators of spine density and morphology [27,28,29]. Arf proteins are divided into three classes based on sequence homology: Class I (Arf1–3), Class II (Arf4–5), and Class III (Arf6). Like other small GTPases, Arfs act as molecular switches that cycle between an active, GTP-bound state and an inactive, GDP-bound state. This cycling activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) [30]. Previous studies have shown that Arf4 mRNA is highly expressed in the postnatal rat DG [31], suggesting that Arf4 might play a role in DG-associated learning and memory tasks. Furthermore, actin filament fragmenting was detected in the rod photoreceptors of transgenic Xenopus laevis expressing a mutant form of Arf4, indicating that Arf4 functions critically in actin cytoskeletal assembly [32]. Since dendritic spines are largely made up of actin, we considered the possibility that Arf4 might be involved in spine development. PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e46340