The Small Gtpase ADP-Ribosylation Factor 6 Negatively Regulates Dendritic Spine Formation

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FEBS Letters 579 (2005) 6834–6838 FEBS 30185

The small GTPase ADP-ribosylation factor 6 negatively regulates dendritic spine formation

Hideyuki Miyazakia,b,1, Masakazu Yamazakia,1,2, Hiroshi Watanabea,3, Tomohiko Maehamac, Takeaki Yokozekia, Yasunori Kanahoa,* a Department of Physiological Chemistry, Graduate School of Comprehensive Sciences and Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575, Japan b Laboratory of Cell and Developmental Biology, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midoriku, Yokohama 226-8501, Japan c Biomembrne Signaling Project, The Tokyo Metropolitan Institute of Medical Sciences, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan

Received 13 September 2005; revised 24 October 2005; accepted 10 November 2005

Available online 28 November 2005

Edited by Jesus Avila

synapses in central nervous system [1], and exhibit morpholog- Abstract Actin cytoskeletal reorganization and membrane trafficking are important for spine morphogenesis. Here we investi- ical plasticity in response to the synaptic activity [2,3]. Mor- gated whether the small GTPase, ADP-ribosylation factor 6 phology of the spine seems to be regulated through actin (ARF6), which regulates actin dynamics and peripheral vesicular cytoskeletal reorganization and membrane dynamics [4,5].It trafficking, is involved in the regulation of spine formation. The has been reported that Rho family small GTPases (Rho, Rac developmental expression pattern of ARF6 in mouse hippocam- and Cdc42) play a crucial role in the regulation of the spine pus was similar to that of the post-synaptic density protein-95, morphogenesis [6–8], probably through reorganization of actin and these molecules colocalized in mouse hippocampal neurons. cytoskeleton. In addition to Rho family GTPases, ADP-ribo- Overexpression of a constitutively active ARF6 mutant in cul- sylation factor 6 (ARF6), a member of ARF family small tured hippocampal neurons decreased the spine density, whereas GTPases, is also involved in actin cytoskeletal reorganization a dominant-negative ARF6 mutant increased the density. These and membrane trafficking [9–13]. These reports raise a possi- results demonstrate a novel function for ARF6 as a key regulator of spine formation. bility that ARF6, as well as Rho family GTPases, regulates 2005 Federation of European Biochemical Societies. Published spine morphogenesis. by Elsevier B.V. All rights reserved. Mammalian ARF family consists of six related gene prod- ucts, ARF1–6, and they are divided into three classes; Keywords: ADP-ribosylation factor 6; Dendritic spine; ARF1–3 are classified into class I, ARF4 and ARF5 into Post-synapse; Actin cytoskeleton class II, and ARF6 is into class III [14,15]. Class I of ARFs, especially ARF1 and ARF3, localize to the endoplasmic reticulum and the Golgi apparatus, and regulate intra-Golgi transport [16–18]. Subcellular localization and physiological function of class II of ARFs are largely unknown. Of three 1. Introduction classes of ARF, the class III of ARF, ARF6, localizes at the plasma membrane and is implicated in the regulation of cor- The small protrusions on dendrites, dendritic spines, in tical actin dynamics and plasma membrane recycling [19].In which actin is enriched, form contact sites for most excitatory the hippocampus, ARF6 mRNA is expressed in CA1–3 pyramidal cell layers and the dentate gyrus [20]. Moreover, it has been reported that overexpression of a dominant-neg- *Corresponding author. Fax: +81 29 853 3271. ative ARF6 mutant (DN) in cultured hippocampal neurons E-mail address: [email protected] (Y. Kanaho). enhances dendritic and axonal branching [21,22], suggesting that ARF6 plays an important role in morphogenesis of hip- 1 These authors contributed equally to this work. pocampal neurons. These reports led us to investigate a pos- 2 Present address: Institute of Molecular Biotechnology of the sible involvement of ARF6 in spine formation in mouse Austrian Academy of Sciences, Dr Bohr Gasse 3-5, 1030 Vienna, Austria. hippocampal neurons. 3 Present address: Department of Molecular Evolution & Genomics, The results obtained in this study provide evidence that Heidelberg Institute of Zoology, University of Heidelberg, Heidelberg, ARF6 negatively regulates the dendritic spine formation. Germany.

Abbreviations: ARF, ADP-ribosylation factor; ARF CA, a constitutively active ARF mutant; ARF DN, a dominant-negative ARF mutant; 2. Materials and methods MAP-2, microtubule-associated protein-2; PIP5K, phosphatidylinositol 4-phosphate 5-kinase; PSD, post-synaptic density; HA, hemagglutinin; 2.1. Antibodies and reagents HRP, horseradish peroxidase; DMEM, DulbeccoÕs modiﬁed EagleÕs An anti-ARF6 antiserum was a generous gift of Dr. J.A. Donaldson. medium; DIV, days in vitro; BSA, bovine serum albumin; FCS, fetal calf The following antibodies and probes were purchased from commercial serum; PBS, phosphate-buﬀered saline; GEF, guanine nucleotide sources: an anti-actin and an anti-microtubule-associated protein 2 exchange factor; P, postnatal days (MAP-2) antibody (SIGMA); an anti-post-synaptic density (PSD)-95

0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.11.022 H. Miyazaki et al. / FEBS Letters 579 (2005) 6834–6838 6835 antibody (Upstate Biotechnology); an anti-hemagglutinin (HA; Roche 3. Results Molecular Biochemicals); a horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Transduction Laboratory); an HRP-conju- 3.1. Expression pattern of ARF6 protein in mouse hippocampus gated anti-rabbit IgG antibody (Amersham); rhodamine phalloidin and Alexa488-conjugated secondary antibodies (Molecular Probes); is similar to that of PSD-95 during development an Cy3-conjugated secondary antibody (Jackson ImmunoResearch To examine a possibility for involvement of ARF6 in hippo- Laboratory) (Fig. 1). campal development, we analyzed the developmental expression profile of the ARF6 protein in mouse hippocampus by 2.2. Generation of Sindbis pseudovirons Western blotting using the antibody specific to ARF6 [24]. Sindbis pseudovirons were generated as previously described [23] Although ARF6 expression was poor in the postnatal days 0 with minor modifications. Briefly, cDNAs for HA-tagged wild type (P0) hippocampus, it gradually increased during development of ARF6 (ARF6 WT-HA), a constitutively active ARF6 mutant (ARF6 CA-HA), a dominant-negative ARF6 mutant (ARF6 DN- until P180 in parallel to that of the post-synaptic density mar- HA), and a constitutively active ARF1 and ARF5 mutants (ARF1 ker protein PSD-95 (Fig. 1A), indicating a link of ARF6 to CA-HA and ARF5 CA-HA, respectively), generous gifts of Dr. K. synaptic function(s). Nakayama, were inserted into the SbaI/StuI site in the pSinRep5 (Invitrogen). Plasmids were linearized and transcribed in vitro. These transcripts and the transcript of DH (26S) helper DNA (Invitrogen) 3.2. Expression and localization of endogenous ARF6 in cultured were co-transfected into Baby Hamster Kidney cells by electropora- hippocampal neurons tion. A Sindbis pseudoviron for EGFP was also prepared by the same To determine whether ARF6 is expressed in hippocampal procedure. After 48 h, the virus particles were harvested and stored at neurons, lysate of 21 DIV hippocampal neurons was subjected À80 C until use. to Western blotting probed with the anti-ARF6 antibody. In this analysis, only single band with an apparent molecular 2.3. Preparation and culture of mouse hippocampal neurons and transient weight of 20 kDa was detected (Fig. 1B), providing evidence expression of ARFs Hippocampi dissected from embryonic day 17 to 18 mice were trea- that ARF6 is expressed in hippocampal neurons and the anti- ted with 10 U/ml papain and 100 U/ml DNase in DulbeccoÕs modified body is very specific to ARF6. EagleÕs medium (DMEM) at 37 C for 20 min. The dissociated cells To determine the subcellular localization of ARF6 in hippo- were then plated onto coverslips coated with poly-L-lysine (100 lg/ campal neurons, hippocampal neurons cultured at the low cell ml) at the density of 250–300 cells/mm2, and cultured in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and density was immunostained for ARF6. Immunostained images 0.5 mM L-glutamine. revealed that ARF6 located in the dendrite, since it colocalized Hippocampal neurons cultured for 21 days in vitro (DIV) were in- with the dendrite-specific marker protein MAP-2 (Fig. 1C). fected with Sindbis pseudovirons and incubated for 16–18 h to ex- Colocalization was evident in 21 DIV neurons (Fig. 1C), prob- press proteins. Under these conditions, about 1% of neurons were ably due to higher expression level of ARF6 protein in 21 DIV infected. neurons than the level in 6 DIV neurons (Fig. 1A). Further- more, it was found that ARF6 in part colocalized with PSD- 2.4. Western blot analysis For Western blot analysis, hippocampal neurons were plated at 95 at the punctate structure of dendrites (Fig. 1D), suggesting 300 cells/mm2, and AraC (1 lM) was added to the culture at 3 the localization of ARF6 at the dendritic spine. However, there DIV. At 21 DIV, neurons were harvested and subjected to Western were a substantial number of ARF6 puncta where PSD95 was blotting as previously reported [23]. In brief, proteins were separated apparently absent. This may be explained by the observation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and reported by Krauss et al. that ARF6 is also enriched in the syn- transferred to polyvinylididene difluoride membranes. The membrane was blocked with the basic buffer consisting of 50 mM aptic plasma membrane of nerve terminal [25]. Tris–HCl, pH 8.0, 2 mM CaCl2 and 80 mM NaCl supplemented with 0.2% Nonidet P-40 and 0.5% bovine serum albumin (BSA), 3.3. Regulation of spine formation by ARF6 and then incubated with primary antibodies. After incubation with the HRP-conjugated secondary antibody, immunoreactive proteins To finally investigate whether ARF6 regulates spine forma- were detected with an ECL immunoblotting detection reagent tion, ARF6 WT, ARF6 CA and ARF6 DN were overexpres- (Amersham Biosciences). sed in hippocampal neurons by the Sindbis virus expression system. In these experiments, actin in neurons was stained to 2.5. Immunofluorescence microscopy and quantification of the density of visualize spines. Interestingly, overexpression of ARF6s drasti- dendritic spines cally affected spine formation (Fig. 2). Overexpression of For immunostaining experiments, cultured hippocampal neurons ARF6 WT significantly decreased the number of spines. The were fixed with 4% formaldehyde in DMEM for 30 min, permeabilized reduction was more prominent when ARF6 CA was overex- with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min, and blocked with 10% fetal calf serum (FCS) and 0.1% bovine BSA in pressed. In contrast, overexpression of ARF6 DN markedly PBS for 1 h. Neurons were then stained by sequential incubation with increased number of spines. primary antibodies in PBS supplemented with 10% FCS and 0.5% Constitutively active mutants of other classes of ARF, ARF1 Tween 20 and secondary antibodies in PBS supplemented with 0.5% CA and ARF5 CA, had little effect, if any (Fig. 3). Thus, effects Tween 20. The immunofluorescently stained cells were then imaged using an fluorescent microscope (Axiovert S100, Zeiss) equipped with of ARF6 on the spine number were very specific. These results, an ORCA-ER CCD camera (Hamamatsu Photonics) and the confocal taken together, provide evidence that ARF6 negatively regulates scanner unit CSU21 (Yokogawa Electric Co.). the spine formation in mouse hippocampal neurons. Spines are defined as actin-rich, stubby and mushroom-shaped protrusions with the ratio more than 2:1 of head diameter to neck diameter emerging from dendrites (see Figs. 2A and 3A). The density of spines per 50 lm dendrite length was quantified by examining 4. Discussion 120–160 separate dendrites from 30 to 40 neurons. The data pre- sented are the means ± S.E. of at least three independent experiments. Statistical significance was evaluated using double-tailed In the present study, we demonstrated that ARF6 negatively StudentÕs t-test. regulates the formation of the dendritic spine in hippocampal 6836 H. Miyazaki et al. / FEBS Letters 579 (2005) 6834–6838

Fig. 2. Effects of ARF6s on the formation of dendritic spines of hippocampal neurons. (A) Twenty one DIV hippocampal neurons were infected with Sindbis pseudovirons to express GFP as a control, ARF6 WT-HA, ARF6 CA-HA and ARF6 DN-HA, and cultured for 16–18 h. Neurons were then immunofluorescently stained for ARF6s (green, left panels) and F-actin (red, middle panels) with the anti-HA antibody/Alexa 488-conjugated secondary antibody and rhodamine phalloidin, respectively. Higher magnifications of images were shown below each panel. Scale bar, 10 lm. (B) Number of spines per 50 lm dendrite length in neurons expressing each protein was quantified. Data represent the means ± S.E. of at least three independent experiments. \P < 0.05.

neurons. It has been reported that spine morphology is regulated through receptors including NMDA receptors [26], brain-derived neurotrophic factor receptors [27,28] and Fig. 1. Developmental expression profile of ARF6 and PSD-95 AMPA receptor subunit GluR2 [29], as well as potassium proteins in hippocampus and subcellular localization of ARF6 in cultured hippocampal neurons. (A) Total lysate (50 lg protein per depolarization [30]. Since the results obtained in this study sug- lane) from mouse hippocampus at various developmental stages gest a novel function of ARF6 as a signaling molecule to reg- were subjected to Western blot analysis probed with the antibod- ulate spine formation, it is of interest to investigate whether ies specific to ARF6 and to the post-synapse density protein PSD- ARF6 is involved in the signaling pathway to regulate the den- 95 as described in Section 2. F-actin was also detected as an dritic spine formation through these receptors. internal control. P, postnatal days. (B) Detection of endogenous ARF6 in hippocampal neurons. Lysate (50 lg protein) from 21 A key question raised here is the molecular mechanism(s) DIV hippocampal neurons was subjected to Western blotting through which ARF6 regulates spine formation. It has been re- probed with the anti-ARF6 antibody as described in Section 2. ported that ARF6 interacts with or regulates several intracel- (C) Immunofluorescent staining of endogenous ARF6 and MAP-2 lular molecules such as arfaptin, phosphatidylinositol 4- in cultured hippocampal neurons. Six DIV (upper panels) and 21 DIV (lower panels) hippocampal neurons were immunostained for phosphate 5-kinase (PIP5K) and phospholipase D1 [14].Of endogenous ARF6 (left panels, green) and MAP-2 (center panels, these downstream effectors of ARF6, PIP5K that reorganizes red) by the anti-ARF6 antibody/Cy3-conjugated secondary anti- actin cytoskeleton through its product phosphatidylinositol body and the anti-MAP-2antibody/Alexa 488-conjugated secondary 4,5-bisphosphate has been reported to be involved in the regu- antibody, respectively. Scale bar, 20 lm. (D) Immunofluorescent lation of the membrane dynamics in non-neuronal cells [31]. staining of endogenous ARF6 and PSD-95. Twenty one DIV hippocampal neurons were immunofluorescently stained for endog- Furthermore, it has been reported that ARF6 fi PIP5K sig- enous ARF6 (red) and PSD-95 (green) by the anti-ARF6 naling pathway is implicated in synaptic vesicle recycling antibody/Cy3-conjugated secondary antibody and the anti-PSD-95 [25], regulated exocytosis [32] and limiting axonal elongation antibody/Alexa 488-conjugated secondary antibody, respectively. and branching [33]. From these reports, it is plausible that Higher magnifications of images were shown below each panel. The lowest panel shows higher magnification of the merged image. PIP5K functions as a downstream effector of ARF6 in the sig- Arrows indicate the colocalized ARF6 with PSD-95. Scale bars, naling pathway for the spine formation. Alternatively, it is 5 lm. possible that the regulation of the spine formation by ARF6 H. Miyazaki et al. / FEBS Letters 579 (2005) 6834–6838 6837

pathways for spine formation. This issue remains to be clariﬁed.

Acknowledgements: The authors are grateful to Drs. Donaldson J.G. and Nakayama K. for providing the anti-ARF6 antibody and the plasmids for ARFs, respectively. This work was supported in part by re- search grants from the Ministry of Education, Science, Sports and Culture, Japan to Y.K.

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