Mol Neurobiol DOI 10.1007/s12035-015-9135-0

Ras Activity Oscillates in the Mouse Suprachiasmatic Nucleus and Modulates Circadian Clock Dynamics Tsvetan Serchov & Antje Jilg & Christian T. Wolf & Ina Radtke & Jörg H. Stehle & Rolf Heumann

Received: 9 December 2014 / Accepted: 22 February 2015 # Springer Science+Business Media New York 2015

Abstract Circadian rhythms, generated in the mouse suprachiasmatic nucleus (SCN), are synchronized to the environmental day–night changes by photic input. The activation of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) and cAMP response element-binding protein (CREB)-mediated transcription play a critical role in this photoentrainment. The small GTPase Ras is one of the major upstream regulators of the ERK1,2/CREB pathway. In contrast to the welldescribed role of Ras in structural and functional synaptic plasticity in the adult mouse brain, the physiological regulation of Ras by photic sensory input is yet unknown. Here, we describe for the first time a circadian rhythm of Ras activity in the mouse SCN. Using synRas transgenic mice, expressing constitutively activated V12-Ha-Ras selectively in neurons, we demonstrate that enhanced Ras activation causes shortening of the circadian period length. We found upregulated expression and decreased inhibitory phosphorylation of the circadian period length modulator, glycogen synthase kinase-3 beta (GSK3β), in the SCN of synRas mice. Conversely, downregulation of Ras activity by blocking its function with

Tsvetan Serchov and Antje Jilg contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9135-0) contains supplementary material, which is available to authorized users. T. Serchov : C. T. Wolf : I. Radtke : R. Heumann (*) Department of Molecular Neurobiochemistry, Ruhr-University, 44780 Bochum, Germany e-mail: [email protected] T. Serchov : R. Heumann International Graduate School of Neuroscience, Ruhr-University, 44780 Bochum, Germany A. Jilg : J. H. Stehle Institute of Anatomy III, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany

an antibody in oscillating cell cultures reduced protein levels and increased phosphorylation of GSK3β and lengthened the period of BMAL1 promoter-driven luciferase activity. Furthermore, enhanced Ras activity in synRas mice resulted in a potentiation of light-induced phase delays at early subjective night, and increased photic induction of pERK1,2/pCREB and c-Fos. In contrast, at late subjective night, photic activation of Ras/ERK1,2/CREB in synRas mice was not sufficient to stimulate c-Fos protein expression and phase advance the clock. Taken together, our results demonstrate that Ras activity fine tunes the period length and modulates photoentrainment of the circadian clock. Keywords Ras . SCN . Photoentrainment . Tau . GSK3β . ERK1,2

Introduction Ras is a universal eukaryotic intracellular membraneanchored 21-kDa protein, which belongs to the large family of small monomeric GTPases. It regulates distinct signal transduction pathways that play an important role in the proper functioning of neurons, where it mediates versatile cellular responses to extracellular cues [1, 2]. In order to study the role of neuronal Ras signaling, we have generated in the past transgenic mice, which express constitutively active V12-Ha-Ras selectively in neurons via a synapsin I promoter (synRas mice), while the endogenous Ras activity continues to be regulated physiologically normal [3]. Previous studies on synRas mice showed a strong increase in Ras/mitogen-activated protein kinase (MAPK) signaling in the cortex and hippocampus [3], resulting in an increased synaptic connectivity [4], an altered adult neurogenesis, and short-term memory deficit

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[5]. While the previous publications characterized Ras as a central regulator of structural and functional synaptic plasticity in the adult nervous system, little is known yet about the physiological regulation of endogenous Ras by sensory input in general and by the day–night changes in environmental lighting in particular. The circadian (circa, about; dies, day) master pacemaker is localized in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus. Sensory input and in particular light is the main resetting stimulus for allowing animals to adjust their biological rhythms to changes in the length of daytime and nighttime [6]. Light exposure during night triggers a cascade of intracellular events that alters the oscillation of the core clock genes and affects clock timing [7]. A welldescribed molecular mechanism for this photoentrainment of SCN is the activation of the MAPK pathway and particularly the extracellular signal-regulated kinases 1 and 2 (ERK1,2) [8–10]. Interestingly, Ras is one of the main upstream regulators of the ERK1,2 signaling route [11], through Ras-binding kinase Raf (MEKK) and MEK1,2 [10, 12]. Light-induced phosphorylation of ERK1,2 is coupled to the activation of transcription factors Elk-1 and cAMP response element-binding protein (CREB) [13–15], thereby triggering the expression of immediate early genes such as c-Fos, Egr1, and JunB [16–19] and notably also the clock genes Per1 and Per2 [20–23]. In vivo studies have shown that inhibition of ERK1,2 in mouse SCN attenuates the phase shifting effects of light [13, 24], CREBdependent transcription, and immediate early gene c-Fos expression [17]. Circadian oscillations are driven by transcription/ translation-based feedback/feedforward loops of a set of clock genes and their protein products [25]. In addition to the transcriptional regulation, posttranslational mechanisms like core clock protein phosphorylation play an important role for plasticity of the SCN clock [26]. Glycogen synthase kinase-3 beta (GSK3β) has a critical function in the fine tuning of the circadian period length [27]. It is one of the upstream kinases, phosphorylating several core clock proteins, such as mammalian BMAL1 [28], CRY2 [29], Rev-erbα, and PER2 [30]. However, it has been shown previously that enhanced Ras signaling increases GSK3β gene expression via the MAPK pathway and ETS2 protein [31]. Furthermore, an increased enzyme activity of GSK3β has been reported previously in synRas mice [32]. Here, we describe a circadian rhythm of Ras activity in mouse SCN. We demonstrate that Ras activation enhances pERK1,2/pCREB signaling and GSK3β activity in the SCN, influencing the light-induced phase resetting of the clock and fine tuning the circadian period length. Therefore, Ras has to be considered as an integral player within the plasticity of mouse circadian clock mechanism.

Material and Methods Animals The establishment of synRas mice has been described earlier [3]. In brief, the 5′-non-translated regions of the human Ha-Ras [33] and the rat synapsin I [34] genes were fused. The 3′-flanking region of the Ha-Ras gene, including its polyadenylation signal, was removed and substituted with a fragment containing internal ribosomal entry site (IRES)/ LacZ. A linear 10.1-kb DNA fragment, without vector sequences, was recovered and was suitable for mouse embryo manipulation. Pronucleus DNA injections and embryo transfers were carried out according to standard procedures. Outbred lines B6CBF1, B6CBF1xMF1, and HimOF1xMF1 were used as embryo donors and recipients. Founders were further bred with lines HimOF1, C57BL/6 (Institute for Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria), and NMRI (Harlan Winkelmann, Borchen, Germany). In particular, line 50 contained one integration site each with multiple transgene integrants. Both were crossed back at least 12 times to NMRI background. Putative homozygous animals of line 50 could not be obtained, and transgenic males were mated with wild-type females for subsequent experiments, renewing regularly the background with young females from Harlan Winkelmann, Borchen, Germany. For all experiments, adult (8–12 weeks of age) wild-type (WT) and transgenic littermates from the synRas mouse line 50 were used. For all experiments, food and water were provided ad libitum.

Locomotor Activity Recordings Animals were housed in individual cages equipped with an infrared logger (Universal Mobile Data Logger, Infra-E-Motion), detecting and recording continuously their horizontal movements. Mice were entrained to a photoperiod of 12:12 h light/dark (LD, lights on at Zeitgeber time (ZT) 0; 400 lx; lights off at ZT12; dim red light 680 nm), for at least 8 days before being transferred to constant darkness (DD), and the locomotor activity was monitored for 20 days consecutively. Light pulses were delivered by applying a monochromatic light source with an intensity of ~400 lx for 15 min at indicated time points, with at least 10 days between individual pulses to allow for consolidation of phase shifts and proper calculation of the timing of the next light pulse. Quantification of the phase shifts and determination of the timing of the next light pulse were performed by ClockLab Software (ActiMetrics Software), where the time difference between regression lines of activity onsets was calculated.

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Tissue Collection and Processing For experiments under LD, mice were sacrificed by cervical dislocation at ZT06 and ZT18. For DD experiments, the mice were kept under dim red light illumination for at least 2 days and then sacrificed at the indicated circadian time (CT) points. Brains were removed and placed in chilled, oxygenated physiological saline (8.1 mM Na2HPO4, 138 mM NaCl, 2.7 mM KCl, and 1.47 mM KH2PO4 [pH 7.4]). For the preparation of the SCN, the brain was coronally cut and the medial optic chiasm was used as an anatomical marker to isolate a cube of tissue from the ventral part of the hypothalamus on both sides of the third ventricle, containing the SCN. The tissue samples were homogenized in an ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 15 mM MgCl2, 10 mM EDTA, 0.1 % SDS, 1 % NP-40, 40 mM NaF, 0.1 % Nadeoxycholate, 1 mM Na-orthovanadate, 1 mM PMSF, and protease inhibitor cocktail [Roche Diagnostics] [pH 7.4]). After clearing the lysate by centrifugation for 15 min at 14,000 g in 4 °C, protein concentrations of supernatants were determined by the Lowry Bio-Rad DC Protein-Assay Kit (BioRad) using BSA as a standard. Anti-Ras-T-24 Cell Line Cultures, Transfection, Serum Shock, and Luciferase Assay The generation and cloning of the anti-Ras-scFv antibody has been performed essentially as described previously [35]. Briefly, the anti-Ras-scFv antibody was cloned from a cDNA library generated from a cell line (Y13-259) expressing antiRas antibody (kindly donated by Dr. Hartmut Land) and the variable regions of the heavy and light chain were linked. The anti-Ras-scFv antibody was C-terminally myc-tagged and subcloned into the TetOff System (BD Biosciences). T-24 cells (ATCC ECV304) [36] were transfected with pTetOff and pBiEGFP-anti-Ras-scFv-myc to generate a stable antiRas-T-24 cell line. Stably transfected cells were cultured in a humidified incubator at 37 °C and 5 % CO2 in RPMI1640 medium containing 10 % fetal calf serum (FCS) supplemented with 50 μg/ml G418, 50 μg/ml hygromycin, and 2 μg/ml doxycycline (all PAA) to suppress anti-Ras-scFv expression. For the induction of the anti-Ras-scFv expression, cells were briefly washed twice with phosphate-buffered saline (PBS) and cultivated in doxycycline-free medium. For the luciferase assay, cells were transfected with 1.5 μg pSGG-ARNTLLuc2P containing BMAL1 promoter luciferase reporter construct (SwitchGear Genomics) with Polyfect transfection reagent (Qiagen), according to manufacturer instructions. Ten hours post-transfection, cells were shifted to serum-free media for 24 h, followed by serum shock at time point 0 by incubation for 2 h in 50 % FCS media. At indicated times, cells were processed for ONE-Glo luciferase assay according to the manufacturer instructions (Promega) or the medium was removed

and cells were washed twice briefly with PBS and lysed in icecold lysis buffer. Inducible expression of anti-Ras-scFv-myc protein was confirmed using Western blot analysis for the presence of the myg-tag (anti-myc 9E10, BD Biosciences). All experiments were performed at least in triplicate. Western Blot Analysis and Ras GTPase Pull-down Assay For Western blot analyses, equal amounts of protein (15 μg) from the SCN lysates were mixed with an equal volume of 2× SDS sample buffer (50 mM Tris–HCL, 12 % sodium dodecyl sulfate (SDS), 20 % 2-mercaptoethanol, 0.8 M DTT, 40 % glycerin, 0.04 % pyronin Y [pH 6.8]) and boiled at 95 °C. Samples were loaded on a 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Maxidouble vertical electrophoresis chamber, Carl Roth GmbH) and transferred to Protran BA83 nitrocellulose membranes (Carl Roth GmbH) by semi-dry electroblotting (Carl Roth GmbH). The membranes were then blocked with 5 % blocking agent (Amersham Biosciences) in Tris-buffered saline and Tween 20 (TBS-T) at room temperature (RT) for 1 h. The blots were then probed with the respective primary antibodies diluted in TBS overnight at 4 °C (anti-Ras 1:10,000, anti-phosphoCREB 1:1000 [Upstate], anti-phosphoERK1,2 [Thr202/Tyr204] 1:2000, anti-phosphoGSK3β 1:1000, antiCREB 1:1000 [all Cell Signaling]; anti-ERK1,2 1:20,000, anti-βIII-Tubulin 1:8000 [all Sigma]; anti-MKP-1 1:1000, anti-c-Fos 1:1000, anti-GSK3β 1:1000 [all Chemicon]). The membranes were washed three times for 10 min in TBS-T and incubated for 1 h at RT with horseradish peroxidase (HRP)conjugated secondary antibody (anti-Mouse IgG-HRP 1:10, 000 [Dianova], anti-Rabbit IgG-HRP 1:2000 [Cell Signaling]), diluted in TBS-T, and then washed again three times for 10 min in TBS-T. The signal was detected using an enhanced chemiluminescence detection (ECL) reagent kit (Amersham Biosciences) as instructed by the manufacturer. To reuse already analyzed blots, the membranes were incubated in stripping buffer (25 mM glycine and 1 % SDS [pH 2.0]) for 1 h at RT and afterwards blocked with 5 % dry milk in TBS-T for 1 h at RT. Subsequent steps were as described previously. All immunoreactive bands obtained in Western blots were quantified densitometrically using the TINA 2.09 software (ISBM) and normalized to total Ras, ERK1,2, CREB, and GSK3β, respectively. In the case of MKP-1, the total ERK1,2 was used for normalization. For measurement of Ras activation, an in vitro pull-down assay was performed by incubating 50 μg protein of SCN lysates along with a GST-tagged Ras-binding domain of Raf-1 kinase (GST-Raf-1-RBD) bound to glutathionesepharose (Amersham Biosciences) for 30 min at 4 °C with intermittent shaking. The mixture was centrifuged at 10,000g for 2 min at 4 °C, and the pellet was washed three times with ice-cold FISH buffer (50 mM Tris–HCl, 100 mM NaCl, and

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2 mM MgCl2 [pH 7.4]) with intermittent centrifugation. After the third centrifugation, the pellet was resuspended in SDS sample buffer, boiled at 95 °C, and loaded on a 12 % SDS– PAGE gel.

Immunohistochemistry Animals were anesthetized with a mix of ketamine-Rompun (ketamine hydrochloride 50 mg and Rompun 0.5 mg per 100 g body weight) (Sigma) and transcardially perfused using an automatic pump (Ismatec, VWR) as described [37]. After perfusion, brains were quickly removed and postfixed overnight in 4 % PFA in PBS and then transferred in 30 % sucrose in PBS for cryoprotection. The brains were embedded in Jung tissue freezing medium (Leica Instruments), and serial sections (40 μm/section) were cut coronally from an ice-cooled block on a sliding microtome HM350 (Microm) through the hypothalamus. The sections were collected and stored in PBS+0.01 % NaN3 (Sigma) at 4 °C for immunohistochemical detection. To determine the ERK1,2 and CREB activation levels, free-floating sections were initially incubated for 10 min with 70 % ethanol and after that incubated for another 10 min with 0.5 % H2O2 in methanol and washed 3× for 10 min with PBS containing 3 % Triton X-100 (PBST). Then the sections were blocked for 30 min with 1 % goat serum in PBST and 1 mM NaF. The brain sections were incubated overnight at RT with an appropriate primary antibody. The tissue was washed six times (5 min/wash) with PBST and incubated with a biotinylated anti-rabbit IgG for 1 h at RT and then with an avidin/biotin HRP complex for 1 h at RT, following the instructions of the manufacturer (ExtrAvidin Peroxidase, Sigma). The signal was visualized by the addition of DAB–nickel-intensified substrate (Vector Laboratories) for 5 min at RT. The sections were dehydrated in 70 %, 80 %, 96 %, and 2× 100 % ethanol and 2× xylol for 3 min each and then mounted on gelatin-coated slides with Permount media (Fisher Scientific). The quantitative analysis of the signal was performed using a Zeiss Axiophot microscope and the IBAS image processing system. Phosphorylation over time was imaged by linear regression using the S-Plus 3.4 software package (MathSoft Inc.). Data from at least three consecutive SCN sections were averaged for each animal analyzed.

Statistical Analysis Data represent the mean±SEM. In all experiments, statistical significance, with p