NEUROREPORT

MOLECULAR NEUROSCIENCE

Inhibition of hippocampal synaptic transmission by impairment of Ral function Bj˛rn Owe-Larssona, Esteban Chaves-Olartec, Ashok Chauhand, Ole Kjaerul¡e, Johan Braska, Monica Thelestamb, Lennart Brodina and Peter L˛wa a

Departments of Neuroscience, bMicrobiology and Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden, cTropical Diseases Research Center, Faculty of Microbiology, University of Costa Rica, San Jose¤, Costa Rica, dDepartment of Neurology, John Hopkins Hospital, Baltimore, Maryland, USA and e Division of Neurophysiology, Department of Medical Physiology, University of Copenhagen, Copenhagen, Denmark. Sponsorship: This work was supported by the Swedish Research Council (projects11287, 4480 and 5969). Correspondence and requests for reprints to Peter L˛w, Department of Neuroscience, Karolinska Institutet; S-17177 Stockholm, Sweden Fax: + 468 325861; e-mail: [email protected] Received1September 2005; revised 8 September 2005; accepted 12 September 2005

Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ras, R-Ras, Rap) both inhibited autaptic responses. In a proportion of the neurons (25%, TcdA-10463; 54%, TcsL-1522), the inhibition was associated with a shift from activity-dependent depression to facilitation, indicating

that the synaptic release probability was reduced. Overexpression of a dominant negative Ral mutant, Ral A28N, caused a strong inhibition of autaptic responses, which was associated with a shift to facilitation in a majority (80%) of the neurons. These results indicate that Ral, along with at least one other non-Rab GTPase, participates in presynaptic regulation in hippocampal c 2005 Lippincott Williams neurons. NeuroReport 16:1805^1808
& Wilkins.

Keywords: Cdc42, exocytosis, hippocampus, Rac, Ral, Rho, synaptic vesicle

Introduction The control of synaptic vesicle cycling is thought to involve small GTPases (guanosine triphosphate) of the Ras superfamily. Among the many members of this family, Rab3 has attracted particular attention. Rab3 is brain-enriched and associated with synaptic vesicles. It interacts with Rim, a component of presynaptic active zones, and with the synaptic vesicle-associated protein rabphilin [1]. Mutation of Rab3 in mouse and Caenorhabditis elegans and overexpression of mutants in different model systems have been found to alter transmitter release [1,2]. Although evidence for a presynaptic role of Rab3 is strong, its precise function remains unclear [1,3]. The related GTPase Rab5 is also present in nerve terminals, in which it is associated with both synaptic vesicles and endosomes. Rab5 has been proposed to participate in synaptic vesicle recycling [4], and in prevention of homotypic fusion between synaptic vesicles [5]. In addition to Rabs, other small GTPases have been implicated in the regulation of synaptic vesicle cycling. Rho, Rac, Cdc42 and Ral are all present in nerve terminals, and the latter two are associated with synaptic vesicles [6–8]. By using large clostridial cytotoxins (LCTs) that inactivate subsets of ras GTPases by monoglucosylation [9], evidence was obtained for involvement of Rac in the presynaptic regulation in Aplysia [8,10]. Furthermore, a presynaptic involvement of Ral has been suggested by studies with mammalian brain synaptosomes [11]. In the present study,

we have used LCTs and protein overexpression to examine whether non-Rab GTPases participate in synaptic transmission in rat hippocampal neurons. Our results suggest that Ral, together with at least one other ras-like GTPase, exerts a prominent regulatory control of transmitter release.

Materials and methods Cell culturing and measurement of toxin uptake The neuron-enriched cultures used to measure toxin uptake were prepared by culturing hippocampal cells in 25 cm2 flasks (Gibco/Invitrogen, Carlsbad, California, USA) for 2 weeks in 10 ml of Neurobasal medium with B27 supplement (1:50), 15 mg/ml gentamicin and 2 mM L-glutamine (NB B27; all from Gibco), which reduces the proportion of glial cells to less than 0.5%. The cells were then treated with Clostridium difficile TcdA-10463 (1.5 mg/ml) for 4–6 h or left untreated. Cells were mechanically removed, resuspended in lysis buffer (50 mM triethanolamine, 150 mM KCl, 2 mM MgCl2, 5 mM guanosine diphosphate, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptine, pH 7.8) and sonicated five times for 5 s. Small GTPases in lysates (200 mg total protein) were labeled in the presence of 30 mM uridine diphosphate-[14C]glucose (NEN Life Science Products Inc., Boston, Massachusetts, USA) with 500 mg/ml of TcdA-10463 for 1 h at 371C. After labeling, proteins were resolved by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and the

c Lippincott Williams & Wilkins 0959- 4965
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NEUROREPORT (b) AMPA response

30 kDa

Protein overexpression with the Semliki Forest virus vector A dominant negative mutant of rat RalA was made by changing serine 28, which corresponds to serine 17 of Ras p21 [14], to asparagine. The quick-change site-directed mutagenesis kit (Stratagene, La Jolla, California, USA) was used with the primer GGCAGTGGTGGTGTGGGCAAGAATGCTCTGACTCTGCAGTTC. The whole open reading frame of Ral was generated with PCR and subcloned into the Bam HI site of the p-Semliki Forest virus (SFV) 1 vector (for details, see Refs [12] and [15]). The infection of hippocampal cultures and determination of whether an individual neuron had been infected was made as previously described [12]. Affinity-purified polyclonal rabbit antibodies to Ral A [16] were used.

Results To perturb small GTPases in hippocampal neurons, we first used two glycosyltransferase LCTs, TcdA-10463, which inactivates Rho, Rac, Cdc42 and Rap, and TcsL-1522, which inactivates Rac, Ral, Ras, R-Ras and Rap [9]. Toxin uptake

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Whole-cell recording Recordings were made at room temperature after replacing the culture medium with a physiological solution [12]. Whole-cell voltage clamp recordings were made from neurons exhibiting a triangular soma of 15–25 mm with distinct dendritic processes [12]. The holding potential was 60 mV. The series resistance was compensated by 75–80%, and was typically 10–15 MO. The neurons, which form synaptic connections onto themselves (so called autapses) under these conditions, were stimulated with a 0.7-ms depolarizing step to + 10 mV [13]. The activitydependent modulation of the autaptic response was tested by stimulation with a train of 50 impulses at 5 Hz; the train was repeated several times [13]. If the ratio of the second/ first response amplitudes exceeded 1, it was classified as facilitating (‘paired-pulse facilitation’). Depression was defined by the same ratio being less than 1. Facilitation was as a rule evident also when the amplitude of subsequent responses in the train was compared with the first response (Figs 1e and 2d). In the sample of control cells (all of which showed depressing responses), the amplitude of the second/first response was approximately 0.5 (Figs 1g and 2f). For statistical analyses, the Student’s t-test or the w2 test was used, as appropriate. Values are given as mean7SEM. In several experiments, the identity of g-aminobutyric acid (GABA)-ergic neurons (see results) was confirmed by applying picrotoxin (Sigma-Aldrich AB, Stockholm, Sweden; 200 mM; seven neurons tested), or by immunolabeling (see below) with a monoclonal antibody to glutamic acid decarboxylase (diluted 1:2000; Roche, Bromma, Sweden).

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glucosylated GTPases visualized by phosphorimager exposition. The mixed hippocampal cultures (i.e. with neurons and glia) used in the electrophysiological experiments were prepared as described previously [12,13]. Ethical permission (N41/04) was approved by The Swedish Animal Welfare Agency. These cultures were used during the third week. Incubation with toxin (TcdA-10463, 1.5–2.5 mg/ml; Clostridium sordelli TcsL-1522, 2.5 mg/ml) was carried out for 4–6 h.

OWE-LARSSON ETAL.

Fig. 1 Inhibition of hippocampal synaptic transmission by large clostridial cytotoxins. (a) Pretreatment of neuron-enriched cultures withTcdA10463 reduces the in-vitro incorporation of [14C]glucose in small GTPases by the same toxin. Lane1shows incorporation in proteins from untreated cells; lane 2 shows incorporation in proteins from toxin-pretreated cells. (b) Mean amplitudes (¢rst response) of glutamatergic autaptic responses in neurons of untreated control cultures (n¼42), and in neurons of cultures treated with TcdA-10463 (n¼6) and TcsL-1522 (n¼21), respectively. (c) Mean amplitudes (¢rst response) of GABAergic autaptic responses under the same conditions as in (b) (n¼42,14 and16, respectively). (d) Sample traces of GABAergic autaptic responses in a control neuron showing depression of the response. The ¢rst and ¢fth responses in a 5 Hz train are shown.The relative postsynaptic current amplitude as compared with the maximum amplitude is plotted below. (e) Sample traces and amplitude plot of GABAergic autaptic responses in a TcsL-1522-treated neuron showing facilitation of the response. (f) Proportion of neurons showing facilitation of the response after treatment with TcdA-10463 and TcsL1522, respectively.The number of non-responding neurons was increased among TcsL-1522-treated neurons (29%; Po0.01) compared with TcdA10463-treated neurons (5%) or controls (7%). (g) Scattered plot diagram of the ratio between the second and ¢rst response for each recorded neuron. GABA, g-aminobutyric acid; AMPA, a-amino-3-hydroxy-5-methyl4 -isoxazole propulonic acid.

into neurons was assayed with TcdA-10463. When neuronenriched cultures had been pretreated with this toxin, subsequent incorporation of uridine diphosphate-[14C]glucose in small GTPases (by in-vitro incubation with the same toxin) was markedly reduced (Fig. 1a). TcdA-10463 is thus effectively taken up into the hippocampal neurons. The effects of the two toxins on synaptic transmission were tested by recording autaptic responses in low-density hippocampal cultures. Responses in these cultures are of two types: short-duration, a-amino-3-hydroxy-5-methyl-4isoxazole propulonic acid receptor-mediated (glutamatergic

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NEUROREPORT

Ral GTPase AND SYNAPTIC TRANSMISSION

Discussion In this study, we have used LCTs and protein overexpression to probe for involvement of Ras-related GTPases in hippocampal synaptic transmission. The protein overexpression experiments identified Ral as a presynaptically acting GTPase. The effect of one of the toxins, TcsL-1522, may be explained, at least in part, by an effect on Ral. The fact that TcdA-10463 also was effective points, however, to the involvement of additional GTPases, such as Rho, Rac, Cdc42 and/or Rap. Rac is a likely candidate as it is associated with synaptic vesicles, and

(a)

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ted ec

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4000 pA

GABA response

Percent showing facilitation

neurons), and long-duration, GABAA receptor-mediated (GABAergic neurons) [12,13,17]. In cultures treated with either TcdA-10463 or TcsL-1522, the amplitude of glutamatergic (Fig. 1b) and GABAergic responses (Fig. 1c) was significantly reduced. To probe for a presynaptic contribution of the effect, we examined the activity-dependent modulation of responses [13]. In control cultures, responses consistently showed depression during the initial phase of a 5 Hz stimulation train (Fig. 1d, f, g). In both TcdA-10463 and TcsL-1522-treated cultures, however, a proportion of the neurons showed facilitating responses (Fig. 1e–g). In these neurons, the amplitude remained at a similar level during the later phase of the stimulus train (TcdA-10463: ratio between the amplitude of the 50th and the first response (50/1) 1.4270.39; ratio 50/10, 1.1570.14; TcsL-1522: ratio of response 50/1, 1.0270.1; ratio 50/10, 0.8770.03 and Fig. 1e). We conclude that toxins inactivating two distinct subsets of GTPases, Rho-Rac-Cdc42-Rap and Ral-Rac-Ras-R-Ras-Rap, respectively, can inhibit hippocampal synaptic transmission, at least partially, by a presynaptic action. To examine the effect of selective GTPase perturbation, we focused on one of the targets of TcsL-1522, Ral. An SFV vector expressing a dominant negative Ral mutant, Ral A28N, was constructed. This mutant prevents activation of Ral (A and B) by forming an inactive complex with Ral GTPase exchange factors [18]. Recordings were made at a time point (12–18 h) when overexpression resulted in intense Ral immunolabeling in the cell body and neurites (Fig. 2a). Uninfected neurons showed almost no labeling and were thus easily distinguished from infected neurons (not shown). Overexpression of Ral A28N had a potent inhibitory action. Both glutamatergic (Fig. 2b) and GABAergic (Fig. 2c) responses were significantly smaller in infected neurons than in uninfected neurons in the same cultures. The activity-dependent modulation showed a marked change, with a majority of the responding neurons exhibiting initial facilitation (Fig. 2d–f). As with the toxins, the amplitude remained at a similar level during the later phase of the train (ratio of response 50/1, 2.2270.83; ratio 50/10, 1.0070.10; Fig. 2d). To verify that the effect of overexpression was specifically linked with the dominant negative mutation, we constructed an SFV vector expressing wild-type Ral (wtRal). As judged from immunolabeling, wtRal showed a similar neuronal distribution and was expressed at a level similar to that of Ral A28N (not shown). Glutamatergic (Fig. 2b) and GABAergic (Fig. 2c) responses were similar in amplitude between wtRal-expressing and uninfected neurons. The activity-dependent modulation in wtRal-expressing neurons was also similar to that in control neurons (Fig. 2e and f).

wtRalA

Fig. 2 Inhibition of hippocampal synaptic transmission by overexpression of a dominant negative Ral mutant. (a) Hippocampal neuron overexpressing Ral A28N detected by immunolabeling with Ral antibodies. (b) Mean amplitudes (¢rst response) of glutamatergic autaptic responses in uninfected and infected neurons of cultures treated with a SFV vector carrying Ral A28N (two left columns; n¼20 and 9, respectively) and wtRal (two right columns; n¼5 and 3, respectively). (c) Mean amplitudes (¢rst response) of GABAergic autaptic responses in the same conditions as in B (n¼39, 11, 19 and 10, respectively). (d) Sample traces and amplitude plot of GABAergic autaptic responses in a Ral A28N-overexpressing neuron showing facilitation of the response. (e) Proportion of uninfected and infected neurons showing facilitation of the response in cultures treated with SFV Ral A28N (two left columns) and SFV wtRal (two right columns), respectively.The number of non-responding neurons did not di¡er statistically between the groups (mean 16%). (f) Scattered plot diagram of the ratio between the second and ¢rst response for each recorded neuron. SFV, Semliki Forest virus; wtRAl, wild-type Ral; GABA, g-aminobutyric acid.

because it has been linked to presynaptic regulation at Aplysia synapses ([8]; see also below). Rho and CDC42 are also candidates as they occur in nerve terminals [8]. Whether Rap is present in nerve terminals is currently unclear, however. To detect presynaptic effects, we monitored the activitydependent modulation during 5 Hz stimulation [13]. This

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NEUROREPORT assay detects inhibitory effects that involve a reduction of the synaptic release probability, which results in reduced depression and enhanced facilitation. It is commonly used to distinguish presynaptic effects from postsynaptic effects (see e.g. [19,13,20]). Recent studies of the presynaptic priming factor munc13 support the validity of this assay [21]. A proportion of the toxin-treated neurons and the majority of the Ral A28N-expressing neurons showed facilitating responses, indicating presynaptic involvement in each case. With regard to TcdA-10463, only a quarter of the neurons showed a shift to facilitation, even though the degree of inhibition was comparable to that induced by TcsL-1522. Although we cannot exclude the fact that TcdA-10463 exerted most of its action postsynaptically, it is interesting to note that Rac inactivation in Aplysia neurons inhibits transmitter release without changing the release probability at individual release sites [8,10]. A role of Ral in synaptic vesicle traffic was first suggested by its association with synaptic vesicles [6,7]. A recent study [11] examined the effect of transgenic expression of Ral A28N on [3H]-glutamate efflux from synaptosomes. The mutant protein caused a reduction of phorbol ester-enhanced efflux, but K + -evoked efflux was unaltered, which appears to be in contradiction with the present findings. One plausible explanation for the discrepancy could be that the levels of Ral A28N in nerve terminals induced by transgenic expression did not fully impair Ral signaling. The SFV system induces very high levels of expression and most of the protein synthesis machinery will express the inserted protein [15]. Another explanation could be that the K + -evoked [3H]-glutamate efflux measured by Polzin et al. [11] was derived partly from non-exocytic sources, such as reversal of plasma membrane transport. While the present work shows that Ral signaling is essential to maintain basal levels of evoked neurotransmitter release, the precise role of Ral in nerve terminals remains unclear. Possible effectors may include many components of the presynaptic machinery, including calcium channels, the actin cytoskeleton and metabotropic glutamate receptors [22,23]. In epithelial cells, Ral has been shown to regulate vesicular transport through interaction with the exocyst complex [24], but this complex may not be involved in neurotransmitter release at synapses [25]. The identification of novel Ral effectors in nerve terminals will thus be of considerable interest.

Conclusion This study has shown that toxins affecting non-Rab GTPases and a dominant negative Ral mutant inhibit hippocampal synaptic transmission, at least in part, by acting presynaptically.

Acknowledgements We thank Dr Christoph von Eichel-Streiber, Johannes Gutenberg-Universita¨t Mainz, for the generous supply of toxins, and Thomas Norlin and Richard Warfvinge for valuable contributions.

OWE-LARSSON ETAL.

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