Charlotte Trautmann

Dominika Burek

Christian A Hübner

Jean-Antoine Girault

PhD Olivia Engmann
email: olivia.engmann@med.uni-jena.de.

A regulatory pathway linking caffeine action, mood and the diurnal clock

1/2,000); β-ACTIN (Sigma,

Introduction

Circadian rhythms govern a large variety of behavioral, physiological and metabolic functions in virtually all cells of the mammalian body. The circadian clock is a complex molecular machinery that drives a large fraction of circadian rhythms. The core clock mechanism is constituted by transcriptional activators CLOCK and BMAL1, which heterodimerize to drive the expression of a large number of clock-controlled genes. The Circadian Locomotor Output Cycles Kaput (CLOCK) protein is a key player in the circadian system, which is crucial for biological time keeping [START_REF] Masri | The circadian clock: A framework linking metabolism, epigenetics and neuronal function[END_REF] and the sleep-wake cycle [START_REF] Archer | How sleep and wakefulness influence circadian rhythmicity: Effects of insufficient and mistimed sleep on the animal and human transcriptome[END_REF]. CLOCK is altered in psychiatric conditions that are characterized by changes in circadian rhythms, including intake of the psychostimulant cocaine (3) and mood disorders [START_REF] Mcclung | Circadian genes, rhythms and the biology of mood disorders[END_REF][START_REF] Mcclung | Role for the Clock gene in bipolar disorder[END_REF].

Caffeine, an antagonist of adenosine receptors, is the most consumed psychostimulant worldwide and has a robust effect on wakefulness. Caffeine can also affect the circadian period of molecular oscillations in vitro [START_REF] Burke | Effects of caffeine on the human circadian clock in vivo and in vitro[END_REF][START_REF] Narishige | Effects of caffeine on circadian phase, amplitude and period evaluated in cells in vitro and peripheral organs in vivo in PER2::LUCIFERASE mice[END_REF] and alters the effects of sleep deprivation in rodents [START_REF] Jha | Sleep Deprivation and Caffeine Treatment Potentiate Photic Resetting of the Master Circadian Clock in a Diurnal Rodent[END_REF][START_REF] Urry | Adenosine, caffeine, and performance: from cognitive neuroscience of sleep to sleep pharmacogenetics[END_REF]. There is accumulating evidence that the psychostimulants cocaine and metamphetamine have an impact on the striatal CLOCK system [START_REF] Falcon | Differential Regulation of the Period Genes in Striatal Regions following Cocaine Exposure[END_REF][START_REF] Uz | Effect of fluoxetine and cocaine on the expression of clock genes in the mouse hippocampus and striatum[END_REF][START_REF] Wongchitrat | Effect of amphetamine on the clock gene expression in rat striatum[END_REF]. However, it is largely unknown how caffeine affects circadian (around the clock) or diurnal (day versus night) signaling. Dopamine-and cAMP-regulated phosphoprotein Mr 32,000 (DARPP-32) has been linked to bipolar disorders, antidepressant action and responses to psychostimulants [START_REF] Svenningsson | DARPP-32: An Integrator of Neurotransmission[END_REF]. Both, adenosine receptors and DARPP-32 are enriched in the medium spiny neurons of the striatum, a main part of the brain reward system. The striatum has previously been shown to be associated with circadian regulation [START_REF] Doi | Impaired light masking in dopamine D2 receptor-null mice[END_REF][START_REF] Yujnovsky | Signaling mediated by the dopamine D2 receptor potentiates circadian regulation by CLOCK:BMAL1[END_REF][START_REF] Spencer | A mutation in CLOCK leads to altered dopamine receptor function[END_REF]. Furthermore, the striatum is involved in a variety of mental illnesses [START_REF] Krishnan | Linking molecules to mood: New insight into the biology of depression[END_REF][START_REF] Mccutcheon | Schizophrenia, Dopamine and the Striatum: From Biology to Symptoms[END_REF][START_REF] Lobo | The striatal balancing act in drug addiction: Distinct roles of direct and indirect pathway medium spiny neurons[END_REF]. Here we describe a previously uncharacterized pathway, which links caffeine response to the diurnal clock system via DARPP-32.

While acute caffeine intake has psychostimulant actions, chronic caffeine affects endophenotypes of depression in mice [START_REF] Yin | Chronic caffeine treatment enhances the resilience to social defeat stress in mice[END_REF][START_REF] Pechlivanova | Effect of long-term caffeine administration on depressive-like behavior in rats exposed to chronic unpredictable stress[END_REF][START_REF] El Yacoubi | Adenosine A2A receptors and depression[END_REF]. Chronic caffeine intake inversely correlated with depression risk in a large longitudinal study [START_REF] Coffee | Caffeine, and Risk of Depression Among Women[END_REF] and depressed patients displayed aberrant circadian synchrony [START_REF] Edgar | Major depressive disorder: A loss of circadian synchrony?[END_REF], with fatigue, mood changes and sleep disturbances being main symptoms of depression [START_REF] Mcclung | Circadian genes, rhythms and the biology of mood disorders[END_REF][START_REF] Lovato | A meta-analysis and model of the relationship between sleep and depression in adolescents: Recommendations for future research and clinical practice[END_REF]. However, it is unclear whether stimulating properties of acute caffeine are intertwined with the CLOCK system at the molecular level [START_REF] Gekakis | Role of the CLOCK Protein in the Mammalian Circadian Mechanism[END_REF] and whether acute administration of caffeine has an impact on mood as well.

Here we demonstrate that DARPP-32 is relevant for the acute mood-elevating actions of caffeine in mice via altering CLOCK-mediated pathways. Caffeine increases the phosphorylation at Thr75 of DARPP-32 [START_REF] Bateup | Cell typespecific regulation of DARPP-32 phosphorylation by psychostimulant and antipsychotic drugs[END_REF][START_REF] Lindskog | Involvement of DARPP-32 phosphorylation in the stimulant action of caffeine[END_REF][START_REF] Engmann | DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons[END_REF]. T75-DARPP-32 reduces CLOCK:Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) binding to chromatin, resulting in caffeine-induced transcriptional changes in the striatum. The effect of DARPP-32 on behavior and chromatin binding of the CLOCK:BMAL1 complex to chromatin is dependent on the light-phase, during which caffeine is administered.

Taken together this study provides a previously unappreciated link between caffeine administration and diurnal signaling and opens new avenues for our understanding of fast-acting effects on mood in the mouse brain.

Materials and Methods

Animals

Luciferase assay

HEK-296 cells were transfected for 24h with luciferase-reporter containing an E-box regulatory element (5 μg), MYC-CLOCK (5 μg), MYC-BMAL (5 μg) and either one of GFP control vector (1 μg), GFP-DARPP-32-wildtype (2 µg) or GFP-DARPP-32-T75A point mutant (2 μg). The plasmid ratios and concentrations have been optimized prior to the experiment to ensure a stoichiometry of luciferase reporter:CLOCK:BMAL:DARPP-32 expression of approximately 1:1:1:1 by using GFP and MYC-antibodies. Total luminescence was normalized to an actin-signal by immunoblotting to account for variations in sample preparation. A standard curve was generated for the total luminescence signal to determine an optimal sample volume (50 μl). The signal from non-transfected lysate was negligible (total luminescence<1). Fifty μl of cell lysate were taken as reaction volume. Total luminescence was normalized against actin and then the average of Luciferase+CLOCK and Luciferase+BMAL1 were subtracted for each of the three groups from the Luciferase+BMAL1+CLOCK group to eliminate background from unspecific binding. Relative luminescence: The average of Reporter+CLOCK and Reporter+BMAL transfections were subtracted from the Reporter+CLOCK+BMAL groups to eliminate background caused by unspecific binding.

Behavioral analysis

Tail suspension test was conducted by taping the mice by their tails to a rod using band aid. The rod was placed approximately 50 cm above a table. Immobility time was measured for 5 min. Forced swim test was performed in a transparent beaker filled with 18°C warm water up to 15 cm below the opening. The time spent immobile was assessed during a 6 min time-period. For novelty suppressed feeding, mice were food restricted for 12 h (app. 1g food per mouse). They were then placed alone in a clean home cage with a food pellet located in the middle of the cage. For a maximum of 10 min, the latency until the mouse starts eating the pellet was recorded. Locomotor activity and anxiolytic behavior were assessed in for 1 h in an open field and analyzed using the TSE Multiconditioning system software (TSE Systems). Numbers of male and female mice were balanced between groups. Data separated by sex are listed in Table S1.

Mice did not undergo the same behavioral paradigm twice. At least one week had to pass between experiments and mice were only receiving caffeine once.

ChIP and quantitative PCR

ChIP was performed essentially as described [START_REF] Engmann | Cocaine-induced chromatin modifications associate with increased expression and 3D looping of Auts2[END_REF]: DNA and proteines were crosslinked using 1% Formaldehyde in PBS for 8 min and the reaction was stopped using 2M glycine. After 4 washing steps with PBS containing EDTA-free protease inhibitor cocktail (Roche, #11873580001), tissue was homogenized by passing it through a 22-gauge needle 30 times. After removing debris (1.230 rcf spin for 5 min, 4°C, discard supernatant), cells were incubated with cell lysis buffer (5mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, Protease inhibitor cocktail) for 15 min and vortexed every 2-3 min. The cells were spun down for 5 min at 2.650 rcf at 4°C and the pellet was resuspended in nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, Protease inhibitor cocktail). They were incubated on ice for 10 min and vortexed every 2-3 min. Next, samples were sonicated in a bioruptor for 2x 12 cycles (30 sec on/off, 5 min break between cycles to avoid overheating). Afterwards, they were spun at 18.000 rcf for 10min at 4°C and the pellet containing chromatin was resuspended in RIPA-buffer (50 mM Tris-HCl, pH 8.0, 5 M NaCl, 2 mM EDTA, pH 8.0, 1% NP-40, 0.5% Sodiumdeoxycholate, 0.1% SDS, protease inhibitors). In parallel, the antibody was coupled to magnetic beads (Dynabeads M-280 Sheep anti-rabbit IgG, ThermoFisher, #11203D).

Per ChIP-reaction, 2 μg of chromatin were incubated with 2 μg of Bmal antibody (Abcam, #ab93806) overnight at 4°C. The dynabeads containing the antibody-chromatin complex were sequentially washed in various buffers to remove unspecifically bound chromatin fragments (1. Low salt buffer: 0.1% SDS, 1% TX-100, 2 mM EDTA, pH 8.0, 150 mM NaCl, 500 mM Tris-HCl, pH 8.0; High salt buffer:0.1% SDS, 1% TX-100, 2 mM EDTA, pH 8.0, 500 mM NaCl, 500 mM Tris-HCl, pH 8.0; 3. LiCl buffer: 150 mM LiCl,1% NP-40, 1% Sodium-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0; 4. TE + 50 mM NaCl: 50 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Beads were collected and chromatin was eluted at 65°C for 30 min (1% SDS, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA). RNA and proteins were removed by digestion with DNAse-free RNAse (Roche, #11 119 915 001) and Proteinase K (Invitrogen, #25520 049) and DNA was purified using the MinElute PCR purification kit (Qiagen, #28004). Concentration and shearing efficiency were assessed with the dsDNA HS Assay and Agilent High-Sensitivity DNA kits (Thermo Fisher Scientific, #Q32851; Agilent Technologies, 5067-4626). Data are represented as ChIP/Input. PCR-primers for Dbp were: Fvd: 5'-ACA CCC GCA TCC GAT AGC-3'; Rev: 5'-CCA CTT CGG GCC AAT GAG-3'. For quantitative PCR, RNA was converted to cDNA using the GoScript Reverse Transcriptase System (Promega, #A5002). During PCR,SsoFast Evagreen reagents were used (Bio-rad, #1725201). The experiment was run on the CFX96 Real-Time System (settings: 1x: 95.0°C, 5 min; 40x: 95°C, 10 sec; 60.0°C, 10 sec; 72.0°C, 10 sec; 1x: 95.0°C, 1 min; 65.0°C, 5 sec; 95.0°C, 5 sec; 4°C). The Per1-signal was normalized with an average of three house keeping genes, Beta-Actin, S18 and Gapdh. The following primers were used: Per1: Fvd: 5'-ACC AGC GTG TCA TGA TGA CAT AC-3', Rev: 5'-GTG CAC AGC ACC CAG TTC CC-3'; β-Actin: Fvd: 5'-TGT TAC CAA CTG GGA CGA CA-3', Rev: 5'-GGG GTG TTG AAG GTC TCA AA-3'; S18: Fvd: 5'-TGG CTC ATT AAA TCA GTT ATG GT-3'; 5'-GTC GGC ATG TAT TAG CTC TAG-3'; Gapdh: Fvd: 5'-AAC TTT GGC ATT GTG GAA GG-3', Rev 5'-ACA CAT TGG GGG TAG GAA CA-3'.

Western blotting und Immunofluorescence

Western blots were performed as described (29): Samples were boiled at 98°C for 10 min in Laemmli buffer (100 mM Tris-HCl, pH 6.8, 12% glycerol 40 g x l -1 SDS, 2% v/v βmercaptoethanol and bromphenol blue). Proteins were blotted using Bio-rad Mini Protean TGX-gels and the Bio-Rad transfer system. Immunofluorescence and quantification for DARPP-32 was performed as described [START_REF] Engmann | DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons[END_REF][START_REF] Engmann | Acute drug-induced spine changes in the nucleus accumbens are dependent on beta-adducin[END_REF]. Essentially, mice were perfused with 4% paraformaldehyde and brains were postfixed overnight at 4°C. They were then cut into 30-μm sections on a Leica vibratome. Free-floating sections were incubated overnight with anti-DARPP-32 antibody at 4°C. After three rinses with TBS, they were incubated with secondary antibody for 2 h at room temperature. After 3 more rinses with TBS, sections were mounted using Fluoromount-G (Southern Biotech, #0100-01). Photos were taken with a Zeiss LSM880 microscope with a 63x magnification. Per brain area, three to four photos per mouse were analyzed.

Statistics

Sample sizes were chosen according to variability estimates in previous experiments or in pilot experiments. Data are expressed as means ± s.e.m. Statistical analysis was performed in GraphPad Prism (version 5). The tests used were two-tailed Student's ttest for comparison of two groups, one-way analysis of variance (ANOVA) when there were more than two groups, two-way ANOVA when two factors were varied and 3-way ANOVA for three factors. When ANOVA was significant, posthoc tests were Newman Keuls-test for one-way ANOVA and Bonferroni test for two-way and three-way ANOVA.

If variances were not equal, Dunn's multiple comparison test was used. Three-way ANOVA data are depicted in Tables S2-S4.

Results

Thr75-DARPP-32 interacts with the C-terminal domain of CLOCK

DARPP-32 is a major hub molecule in striatal medium spiny neurons. Previously, LC-MS/MS on striatal lysate pulled down from purified DARPP-32 showed the Acid tolerance regulated protein, which is highly homologous to CLOCK [START_REF] Doi | Circadian Regulator CLOCK Is a Histone Acetyltransferase[END_REF], as potential binding partner [START_REF] Engmann | DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons[END_REF]. Hence, we hypothesized that DARPP-32 may be able to bind to CLOCK as well. Therefore, MYC-CLOCK and DARPP-32-GFP were co-expressed in COS7-cells and cell lysates were subsequently incubated on a MYC-affinity column [START_REF] Engmann | DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons[END_REF]. DARPP-32 was pulled down with CLOCK (Fig. 1). The experiment was repeated with MYC-BMAL1 and DARPP-32-GFP-CLOCK, however no signal for DARPP-32 was detected in the pulldown. DARPP-32 is a phospho-protein with a variety of molecular functions that depend on its phosphorylation state on four residues [START_REF] Yger | DARPP-32, Jack of All Trades? Master of Which?[END_REF]. In order to determine the protein domains of DARPP-32 implicated in CLOCK-binding, COS-7 cells were transfected with a CLOCK plasmid and a variety of truncated and mutated forms of DARPP-32. CLOCK-binding was abolished in DARPP-32 Cter1, which lacks the N-terminal amino acids aa1-91 of the protein including the regulatory phosphosite Thr75. Therefore, a T75A mutant form of DARPP-32 was tested for CLOCK binding as well. Remarkably, the T75A mutation prevented DARPP-32 binding to CLOCK (Fig. 1A-D).

Next, we sought to identify the domain of CLOCK involved in DARPP-32 binding.

Various CLOCK mutant plasmids were co-transfected with full length DARPP-32 and the pulled down fractions were analyzed by western blot using an anti-DARPP-32 specific antibody. While truncated versions of CLOCK containing aa1-625 and aa1-570 did not impact on CLOCK-DARPP-32 interaction, DARPP-32 binding did not occur on the CLOCK mutant lacking a C-terminus ("DN"; lacking the N-terminal amino acids 1-269) (Fig. 1F-G) [START_REF] Doi | Circadian Regulator CLOCK Is a Histone Acetyltransferase[END_REF]. Taken together, these data suggest that DARPP-32 interacts with CLOCK in its C-terminal region in vitro and that this process is dependent on Thr75-DARPP-32.

To assess whether DARPP-32 and CLOCK interact in vivo, we performed coimmunoprecipitation (Co-IP) using DARPP-32-GFP transgenic mice versus WT littermates and used an anti-CLOCK specific antibody for western blot quantification.

GFP-pulldowns from these mice showed a significant enrichment of CLOCK, suggesting that CLOCK can interact with DARPP-32 in the striatum in vivo (Fig. 1I,J).

Thr75-DARPP-32 reduces activation by the CLOCK:BMAL1 complex

The CLOCK:BMAL1 complex activates transcription via binding chromatin at E-box consensus sites [START_REF] Gekakis | Role of the CLOCK Protein in the Mammalian Circadian Mechanism[END_REF]. In order to determine whether the presence of DARPP-32 may disrupt CLOCK:BMAL1-binding to the E-box, luciferase assays were performed in HEK-293 cells transfected with CLOCK, BMAL1, a luciferase reporter and various forms of DARPP-32 or control (GFP) vectors. The activity of the reporter was much higher when CLOCK and BMAL1 were co-transfected than when either one was transfected alone (Fig. S1). Importantly, and in agreement with the Co-IP data (Fig 1A-D), DARPP-32 significantly reduced luminescence, suggesting reduced binding of CLOCK:BMAL1 to the E-box (Fig. 1K). DARPP-32 reduced luminescence only when both CLOCK and BMAL1 were present. The T75A mutation in DARPP-32 completely abolished this effect.

Hence, at least under these artificial in vitro conditions, Thr75-DARPP-32 alters CLOCK:BMAL1 binding to chromatin and subsequent gene expression.

Caffeine affects CLOCK:BMAL1 binding to chromatin and gene expression via Thr75-DARPP-32 in vivo

DARPP-32 phosphorylation on Thr75 is increased by caffeine [START_REF] Lindskog | Involvement of DARPP-32 phosphorylation in the stimulant action of caffeine[END_REF][START_REF] Engmann | DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons[END_REF]. Since Thr75-DARPP-32 can reduce CLOCK:BMAL1 binding to chromatin in vitro, we hypothesized that this effect may also be observed in vivo in response to administration of caffeine.

WT mice were IP-injected with caffeine at four time points along the day and night-cycle.

Striatal tissue was collected 40 min later when Thr75-phosphorylation on DARPP-32 is known to be at a maximum [START_REF] Lindskog | Involvement of DARPP-32 phosphorylation in the stimulant action of caffeine[END_REF]. Striatal lysates were chromatin-immunoprecipitated with an antibody against BMAL1. D site of albumin promoter binding protein (Dbp) is a canonical CLOCK:BMAL1 target gene and therefore we measured Dbp by PCR and used Dbp-pulldown by BMAL1 as a quantitative readout. In both saline and caffeine conditions, diurnal oscillations of pulled-down Dbp-levels paralleled those previously reported [START_REF] Koike | Transcriptional architecture and chromatin landscape of the core circadian clock in mammals[END_REF]. Importantly, across the entire day and night-cycle, caffeine significantly reduced Dbp-pulldown (Fig. 2A).

To investigate, whether the effect of caffeine on CLOCK:BMAL1 signaling is dependent on Thr75-DARPP-32, the experiment was repeated with T75A-DARPP-32 knock-in mutant mice, in which caffeine-dependent Thr75-phosphorylation is prevented [START_REF] Engmann | DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons[END_REF].

Dbp-pulldown under baseline conditions was not significantly altered in T75A-DARPP-32 knock-in mice (Fig. 2B). However, while in WT mice, caffeine reduced CLOCK:BMAL1 binding to Dbp in the active phase of the light cycle, this effect was abolished in T75A-DARPP-32 mutants. (Fig. 2C) During the light (inactive) phase, the T75A-mutation did not affect binding to the Dbp-gene (Fig. 2D). Similarly, caffeine reduced gene expression of the circadian regulator Period 1 (Per1) 2 h post-injection in WT but not T75A-mutants during the active phase. In agreement with the data above, the mutant effect on caffeine-induced Per1-expression did not occur in the light phase (Fig. 2E,F). DARPP-32 can translocate into the nucleus upon phosphorylation of the Ser97-site [START_REF] Stipanovich | A phosphatase cascade by which rewarding stimuli control nucleosomal response[END_REF] and may contribute to the presence of DARPP-32 in the vicinity of CLOCK in vivo.

Therefore we tested whether DARPP-32 translocation occurred in a diurnal manner. However, this was not the case (Fig. S2). Additionally, we did not find evidence for diurnal changes in Thr75-phosphorylation (Fig. S3). It is therefore unlikely that the effects of DARPP-32 on CLOCK:BMAL1 binding are due to diurnal fluctuations in nuclear phospho-Thr75-DARPP-32.

Mood-elevating effects of caffeine are prevented by T75A-DARPP-32 mutation in a diurnal manner

Since chronic caffeine has been shown to have positive effects on depressive-like behaviors in rodents, we asked whether caffeine in non-depressed may positively affect mood-related behaviors as well and whether T75-DARPP-32 might be involved in this effect.

WT mice and T75A mutants were injected with caffeine and assessed for behaviors typically linked to antidepressant responses or mood 40 min and 2 h later. Since neither WT mice not T75A mutants can be considered depression models under normal circumstances, an improvement in such behaviors may be termed mood-elevating.

Escape behavior was measured in tail suspension and forced swim tests given their temporal specificity. Since the chromatin-IP-data suggested that the light phase is relevant for the effect of the T75A mutation, behavioral tests were conducted in both, light-and dark phases. During the dark phase, 40 min of caffeine efficiently reduced the relative immobility time in WT mice, both in forced swim and tail suspension tests (Fig. 3A,B). This effect was abolished in T75A-DARPP-32 mutants. Furthermore, caffeine had similar T75-dependent effects in the novelty-suppressed feeding test (Fig. 3C,).

During the light phase, 40 min of caffeine significantly reduced escape behavior in the tail suspension test (Fig. 3D), while only having a very mild or no effect in the forced swim test and novelty suppressed feeding (Fig. 3E,F). Importantly, the effects of caffeine were indistinguishable between WT mice and T75A-DARPP-32 mutants (Fig.

3D-F).

When all factors (light phase, drug and genotype) were compared, in the forced swim test, 3-way ANOVA revealed a significant interaction of light phase, drug and genotype (Table S2). The Tukey posthoc test showed significant effects of the drug within WT in the dark phase, and of the genotype within the caffeine-group during the dark-phase (Table S2). During tail suspension and novelty suppressed feeding tests, 3-way ANOVA did not reveal a significant interaction of circadian time, drug and genotype (Tables S3,S4). However, other comparisons were significant such as effects of genotype, and drug, for the tail suspension test and light phase, and drug, for novelty suppressed feeding. For the tail suspension test, the Tukey posthoc test again showed significant effects of the drug within WT in the dark phase and of the genotype within the caffeinegroup during the dark-phase (Table S3). For novelty suppressed feeding these measures were not significant, however there were significant effects of light phase within T75A-mutants and of drug within WT. Taken together, these results support the observation that caffeine effects depend on the T75-DARPP-32 mutation and the light phase.

The T75-dependent effects of caffeine in the dark phase measured in tail suspension, forced swim and novelty suppressed feeding tests persisted at the 2 h time point in a new cohort of mice (Fig. 3 G-I), suggesting that behavioral effects persist at a time point, when gene expression changes occur (Fig. 2).

Notably, while caffeine increased the locomotor activity as expected, it did so equally in WT and T75A littermates (Fig. S4). Hence, the effects on escape behavior are unlikely to be due to locomotor effects of caffeine. Furthermore, while caffeine increased anxiolytic behavior during the active phase (Fig. S4), it did so equally in WT and T75Amutant mice. Taken together, these data support an interaction between the caffeinephospho-Thr75-DARPP-32 pathway predominantly in the active phase of the day and night-cycle and suggest effects on molecular and behavioral levels.

Discussion

Depression and circadian / diurnal rhythms have been linked. Here we reveal a regulatory pathway which connects caffeine action to CLOCK:BMAL1 signaling via Thr75-DARPP-32. Thr75-DARPP-32 directly binds to CLOCK and disrupts CLOCK:BMAL1 binding to chromatin, subsequent gene expression, and mediates mood-elevating effects of caffeine. This effect is dependent on the light phase, encouraging that future pharmacological studies are being conducted at specific circadian or diurnal time points.

While we show that Thr75 is relevant for the interaction between DARPP-32 and CLOCK, a role of Thr75 phosphorylation is not formally demonstrated. It is a likely explanation but alternatively Thr75 may be necessary for the interaction regardless of phosphorylation state. To test these two scenarios, purified DARPP-32 that has been phosphorylated with cdk5 in vitro (versus control) may be used for the co-immunoprecipitation experiments in the future. Alternatively, a Threonine to glutamic acid mutation could be used as a phospho-mimetic.

We observed that DARPP-32 binds to the C-terminal domain of CLOCK, which is necessary for heteromerization with BMAL1. There are various scenarios by which DARPP-32 may affect CLOCK, BMAL1 or chromatin, resulting in altered CLOCK:BMAL1 binding: First, DARPP-32 can inhibit protein phosphatase-1 and protein kinase A. In consequence, phosphorylation states of CLOCK and/or BMAL1 may be altered. Second, DARPP-32 may affect histone phosphorylation, for instance on histone 3 Serine 10 (31), which may directly or indirectly affect CLOCK:BMAL1-binding. Third, DARPP-32 may sterically obstruct BMAL1 from CLOCK-binding. Further experiments are warranted to explore the exact mechanisms, by which DARPP-32 affects CLOCK:BMAL1, particularly in vivo. Since T75A-mutation abolished DARPP-32 binding to CLOCK:BMAL1 in luciferase essays (Fig. 1K) but did not affect chromatin binding in ChIP with BMAL1antibodies (Fig. 2B), additional binding partners and pathways may be relevant in vivo.

The presented experiments explore the in vivo effects of this pathway mostly from the angle of DARPP-32. More studies on the effect of CLOCK and BMAL1 in this respect may provide further support. This includes a thorough assessment of properties in CLOCK:BMAL1 occupancy on chromatin in the T75A-DARPP-32 mouse model, such as period of oscillation, levels and peak phases of various CLOCK-regulated genes. Since this study was done in a 12D/12L light cycle, further experiments under various other light conditions may exclude that the observed effects are diurnal rather than circadian.

Similarly, it should be investigated whether the DARPP-32:CLOCK pathway is also contributing to the effect of caffeine on the circadian period.

The main target of caffeine in the brain are adenosine receptors. These are expressed in various brain regions, including forebrain, striatum and hippocampus. It is well established that Thr75 phosphorylation on DARPP-32 occurs via adenosine A2Areceptors [START_REF] Hsu | Caffeine and a selective adenosine A2A receptor antagonist induce reward and sensitization behavior associated with increased phospho-Thr75-DARPP-32 in mice[END_REF][START_REF] Shen | Adenosine a2A receptors in striatal glutamatergic terminals and GABAergic neurons oppositely modulate psychostimulant action and DARPP-32 phosphorylation[END_REF]. Additionally, caffeine has pleiotropic effects beyond striatal adenosine-receptor binding.

Since the T75A-mutation blocks chromatin binding of CLOCK:BMAL1, caffeine likely affects gene expression through the A2A-R:Thr75-DARPP-32 pathway in the striatum.

Since A2A-receptors in either forebrain or striatum can affect Thr75 phosphorylation [START_REF] Shen | Adenosine a2A receptors in striatal glutamatergic terminals and GABAergic neurons oppositely modulate psychostimulant action and DARPP-32 phosphorylation[END_REF], it is not clear, whether the analysed striatal DARPP-32 is regulated by A2Areceptors within the striatum, outside the striatum and is affected additionally by other caffeine-responsive pathways.

T75A-mutation also blocks behavioral effects induced by caffeine. DARPP-32 is most strongly expressed in the striatum. Hence, it is likely that the observed striatal signaling plays a strong role in the observed mood elevating effects. However, since DARPP-32 is also expressed in other tissues, albeit at lower levels, at this point we cannot exclude that other brain areas have an effect on the behavioral outcome as well.

While we used Dbp pulldown and Per1 gene expression to demonstrate the relevance of DARPP-32 to CLOCK:BMAL1 chromatin binding, in the future, chromatin binding and gene expression profiles should be analyzed in a more unbiased and extensive way to better understand the molecular consequences of the described pathway.

Clinical studies on caffeine in depressed cohorts are scarce and hence, too few data are available to draw conclusions regarding the (fast-acting) antidepressant potential of caffeine in humans. However, in rodent studies caffeine does have reproducible effects on depressive-like behavior or mood in various paradigms. Nevertheless, we would like to emphasize that caffeine was used in this study as a tool to identify novel pathways that may rapidly improve mood. Our aim is not to suggest using caffeine itself as an antidepressant or mood elevator, given its already widespread use in the population as well as potential side effects. The behavioral tests in the study presented here should be repeated in a depression-model such as chronic variable stress [START_REF] Labonte | Sex-Specific Transcriptional Signatures in Human Depression[END_REF] to assess, whether the mood-elevating effects of acute caffeine can be beneficial in depressive-like states as well. Moreover, determining a time course of caffeine-induced effects and comparing acute and chronic consequences on molecular and behavioral alterations will be important goals for the future. Nevertheless, ultimately extensions of our study may help identifying potential molecular drug targets for fast-acting antidepressant effects or mood elevation.

Conclusions

Caffeine can affect CLOCK:BMAL1 binding to chromatin via Thr75-DARPP-32. This pathway mediates, at least in part, fast-acting mood-elevating effects of caffeine in mice.

Figure legends

  Mice were housed in accordance with the ethical guidelines (Declaration of Helsinki and NIH, publication no. 85-23, revised 1985; the European Community Guidelines; and the French Agriculture and Forestry Ministry guidelines for handling animals, decree 87849, license A 75-05-22) and approved by the Institut du Fer à Moulin ethical committee. Furthermore, experiments and animal maintenance were conducted in compliance with the Federation of Swiss Cantonal Veterinary Office and approved by the Zürich Cantonal Veterinary Office under license ZH241/16. They were also approved by the Thüringer Landesverwaltungsamt, Germany, under License UKJ-18-036. Male C57Bl/6J mice were purchased from Janvier. DARPP-32 T75A knock-in mutants (PPP1R1B-T75A) (30) and DARPP-32-GFP BAC transgenic mice (29) were kindly provided by Paul Greengard, The Rockefeller University, New York. Male and female mice were used at 6-18 weeks of age and pooled in equal numbers for experiments. All behavioral experiments were conducted in a 12D:12L light cycle. Behavioral experiments during the dark phase were conducted in red light.

Fig. 1

 1 Fig. 1 Thr75-DARPP-32 binds to the C-terminus of CLOCK and disrupts E-box binding. A-H) COS7-cells were co-transfected with MYC-CLOCK and DARPP-32-constructs and cell lysates loaded on a MYC-affinity column. A-D) DARPP-32 binding to CLOCK requires Thr75. A) DARPP-32 constructs. B) Western blot of input for the Co-IP. C) Western-blot of the pulldown. D) Quantification of the pulldowns. Pulldown was indistinguishable from the GFP-control when the DARPP-32 C-terminus (aa 1-91), including Thr75, was deleted or when Thr75 was mutated to alanine (T75A). n = 7-20 per group, Kruskal-Wallis test: P < 0.001, Dunn's Multiple Comparison-Test: WT vs Cter1: **P < 0.01, WT vs. T75A: **P < 0.01; Cter vs. T75A: P > 0.05. E-H) CLOCK binds DARPP-32 via its C-terminal domain. e) Overview over CLOCK constructs. F) Western blot of input for the Co-IP. G) Western-blot of the pulldown. H) Quantification of the pulldowns. DARPP-32 binding did not occur on the "DN" mutant, which is lacking the Cterminus of CLOCK. n = 8-20 per group, Kruskal-Wallis test: P < 0.05, Dunn's multiple comparison test: DN vs all other groups: *P < 0.05; all other comparisons: P > 0.05. I, J) CLOCK and DARPP-32 bind to one another in vivo. Striatal lysates from DARPP-32-GFP-knock-in mice and WT littermates were immunoprecipitated with GFP antibodies. I) Western-blot of the pulldown. J) Quantification of the pulldown. n = 10,12 per group, Student's t-test: t(20) = 2.13, *P < 0.05. K) WT-DARPP-32 but not T75A, reduces CLOCK:BMAL1-binding to an E-box reporter in a luciferase-assay. n = 6 per group; One-way ANOVA: P < 0.0001, Newman-Keuls posthoc test: DARPP-32 vs. GFP: ***P < 0.001; DARPP-32 vs. T75A: ***P < 0.001. SM: size marker. Means +/-s.e.m. and individual data points are shown.

Fig. 2

 2 Fig. 2 Caffeine reduces CLOCK:BMAL1 binding to chromatin and gene expression in WT but not T75A-DARPP-32 mutants. A, C-F) Caffeine, 7.5 mg/kg, was intraperitoneally (IP)-injected at different time points along the circadian cycle. Striatal tissue was extracted 40 min later and chromatinimmunoprecipitated with a BMAL1-antibody. Dbp-pulldown was quantified by RT-PCR. A) Dbp-pulldown is reduced by caffeine. n = 15-16 per group; 2-way ANOVA: effect of time: F(3,116) = 2.85, P < 0.05; effect of caffeine: F(1,116) = 13.97, ***P < 0.001. B) Dbppulldown is not significantly affected by T75A-mutation under baseline conditions. n = 8-14 per group; 2-way ANOVA: effect of time: F(3,79) = 2.94, P < 0.05. Bonferroni posthoc test: WT vs. T75A for all time points: P>0.05. C) During the dark (active) phase (ZT 12-24), caffeine reduces Dbp-pulldown in WT but not T75A-mutants. n = 8-10 per group; 2way ANOVA: interaction between caffeine and genotype: F(1,33) = 3.19, P = 0.08; Bonferroni posthoc test: effect of caffeine in WT: *P < 0.05, in T75A: P > 0.05. D) During the light (inactive) phase (ZT 0-12), caffeine-induced reductions in Dbp-pulldown are indistinguishable between WT and T75A-mutants. n = 9-14 per group; 2-way ANOVA: interaction between caffeine and genotype: F(1,44) = 0.14, P > 0.05; Bonferroni posthoc test: effect of caffeine in WT: P > 0.05; in T75A: P > 0.05. E) During the dark phase, caffeine reduces Per1 gene expression in WT but not T75A-mutants. n = 5-7 per group; 2-way ANOVA: effect of caffeine: F(1,24) = 6.339, P < 0.05; Bonferroni posthoc test: effect of caffeine in WT: *P < 0.05, in T75A: P > 0.05. F) During the light phase, caffeineinduced reductions in Per1 expression are indistinguishable between WT and T75Amutants. n = 5-6 per group; 2-way ANOVA: all measures: F(1,22) < 1.74, P > 0.05. Means +/-s.e.m. and individual data points are shown. ZT = Zeitgeber time; FC = fold change.

Fig. 3 Fig. 3

 33 Fig. 3 Mood-elevating effects of caffeine during the active phase are prevented by T75A-DARPP-32 mutation. A-F). WT mice and T75A mutant were acutely injected with saline vs. caffeine and tested 40 min later in depression-related behavioral paradigm. A-C) The percentage of time spent immobile was assessed in the dark phase of the circadian cycle (ZT 12-24). A) Tail suspension test: n = 6-9; 2-way ANOVA: Effect of genotype: F(1,26) = 16.05, P < 0.05; Effect of caffeine: F(1,26) = 17.34, P < 0.01; Bonferroni Posthoc-test: Effect of caffeine in WT: **P < 0.01; in T75A: P > 0.05. B) Forced swim test: n = 6-8; 2-way ANOVA: Interaction between genotype and caffeine: F(1,24) = 8.794, P < 0.01; Bonferroni Posthoc-test: Effect of caffeine in WT: ***P < 0.001, in T75A: P > 0.05. C) Novelty suppressed feeding: n=6-10; 2-way ANOVA: Effect of caffeine: F(1,30) = 6.00, P < 0.05; interaction between genotype and caffeine: F(1,30) = 5.30, P < 0.05; Bonferroni Posthoc-test: Effect of caffeine in WT: **P < 0.01, in T75A: P > 0.05. D-F) Same measures as A-C but in light phase (ZT 0-12). D) Tail suspension test: n = 7-12; 2-way ANOVA: Effect of caffeine: F(1,32) = 19.70; Bonferroni Posthoc-test: Effect of caffeine in WT: **P < 0.01; in T75A: **P < 0.01. E) Forced swim test: n = 7-9; 2-way ANOVA: Effect of caffeine F(1,27) = 4.218; P < 0.05; Bonferroni Posthoc-test: Effect of caffeine in WT: P > 0.05, in T75A: P > 0.05. C) Novelty suppressed feeding: n = 7-9; 2-way ANOVA: all effects n.s. G-I) Behavioral tests were conducted in the dark phase (ZT 12-24) 2 h after injection of saline vs. caffeine. G) Tail suspension test: n = 5-8; 2-way ANOVA: Effect of drug: F(1,23) = 8.32, P < 0.01; Interaction between caffeine and genotype: F(1,23) = 12.22, P < 0.01; Bonferroni Posthoc-test: Effect of caffeine in WT: ***P < 0.001; in T75A: P > 0.05. H) Forced swim test: n = 6-10; 2-way ANOVA: Effect of drug: F(1,28) = 4.79, P < 0.05; Bonferroni Posthoc-test: Effect of caffeine in WT: **P < 0.01, in T75A: P > 0.05. I) Novelty suppressed feeding: n = 12-13; 2-way ANOVA: Effect of caffeine: F(1,46) = 4.69, P < 0.05. Interaction between genotype and caffeine: F(1,46) = 22.29, P < 0.001; Bonferroni Posthoc-test: Effect of caffeine in WT: *P < 0.05, in T75A: P > 0.05. Means +/-s.e.m. and individual data points are shown.

Acknowledgements

We thank Prof. Isabelle M. Mansuy (University of Zurich, ETH Zurich, Switzerland) and Prof. Eric J. Nestler (Mount Sinai School of Medicine, USA) for their generous support in providing lab space.

Funding and disclosure

This project has been supported by a NARSAD Young Investigator grant (#23661) and an Advanced Medical Scientist Award from the Interdisciplinary Center for Clinical Research (IZKF) at the Universitätsklinikum Jena, Germany (#973685). JAG was supported by ERC-2009-AdG and Fondation pour la recherché médicale.

The authors declare no competing interests.

Author contributions


Ni An
email: ni.an@enpc.fr

Yu-Jun Cui

Nathalie Conil

Jean Talandier

Sebastien Conil

Soil-atmosphere interaction in the overburden of a short-lived low and intermediate level nuclear waste (LLW/ILW) disposal facility

Keywords: Soil-atmosphere interaction, Soil hydro-thermal behavior, Further meteorological information, Different time scales, Long-term estimation

Abstract:The long-term safety of shallow disposal facility for short-lived low and intermediate level nuclear waste (LLW/ILW) relies on the performance of engineered barriers (caps and liners).

Soil-atmosphere interaction may affect the capping material at shallow depths, due to changes of its coupled hydro-thermal-mechanical behavior under the effect of atmospheric conditions. This study aims to investigate the soil-atmosphere interaction at different time scales in the overburden of an LLW/ILW disposal facility, located in middle France. Actual meteorological data from Valencia El Saler station in Spain, including solar radiation, air temperature, latent heat, rainfall, and actual evaporation, at four different time scales (30 min, daily, weekly and monthly), were applied as the future climate conditions of the studied area based on climate analogues. A numerical approach combining a coupled hydro-thermal model and a soil-atmosphere interaction model was employed. Results show that the employment of meteorological data at a short time scale (30 min) can increase the simulation accuracy by capturing the extreme climate events and accelerating the soil-atmosphere interactions. Furthermore, the effect of future climate conditions in the long-term (7 years) on soil hydro-thermal behavior was examined. This enables a further detailed inspection of the climate's role in the LLW/ILW storage system.

Introduction

Nuclear waste is currently being disposed at a number of facilities all over the world [START_REF] Giusti | A review of waste management practices and their impact on human health[END_REF][START_REF] Mccartney | Energy geotechnics: Advances in subsurface energy recovery, storage, exchange, and waste management[END_REF][START_REF] Zhang | Reliability analysis of deep underground research laboratory in Beishan for geological disposal of high-level radioactive waste[END_REF].

Considering the decay of radioactivity, a near-surface type of disposal facility is usually proposed for the short-lived low and intermediate level nuclear waste (LLW and ILW) [START_REF] Yim | Performance assessment models for low level radioactive waste disposal facilities: a review[END_REF]. By contrast, the solution for the disposal of high-level and other long-lived nuclear waste (HLW) is a deep geological repository [START_REF] Mccartney | Energy geotechnics: Advances in subsurface energy recovery, storage, exchange, and waste management[END_REF][START_REF] Kim | A multi-objective optimization of initial conditions in a radioactive waste repository by numerical thermo-hydro-mechanical modeling[END_REF]. In the case of LLW/ILW repository, soil-atmosphere interaction may affect its capping material at shallow depths by the physical process of heat and mass transfer over time. For instance, cracks may be produced as a consequence of long dry and hot periods, significantly modifying the thermo-hydro-mechanical properties of the capping material; contaminant transport in the host geologic formation may be accelerated due to changes in soil temperature, water content and suction from weathering, etc. [START_REF] Näslund | Climate considerations in long-term safety assessments for nuclear waste repositories[END_REF][START_REF] Vardon | Climatic influence on geotechnical infrastructure: A review[END_REF]. Hence, the effect of climate change must be considered in the assessment of the long-term performance of LLW/ILW disposal.

Soil-atmosphere interaction drives water, energy and biogeochemical cycles, causing variations of soil moisture, temperature, and suction, etc. [START_REF] Blight | Interactions between the atmosphere and the Earth[END_REF][START_REF] Cui | Experimental and numerical investigation of soil-atmosphere interaction[END_REF][START_REF] An | Numerical analysis of soil volumetric water content and temperature variations in an embankment due to soil-atmosphere interaction[END_REF]. Over recent years, increasing awareness about the importance of soil-atmosphere interaction has led researchers to investigate the potential effect of climate change on the performance of waste covers and nuclear waste handing [11][START_REF] Van Geet | Climate evolution in the long-term safety assessment of surface and geological disposal facilities for radioactive waste in Belgium[END_REF][START_REF] Fischer | Hydrogeologic factors in the selection of shallow land burial sites for the disposal of low-level radioactive waste[END_REF][START_REF] Fischer | Sediment properties and water movement through shallow unsaturated alluvium at an arid site for disposal of low-level eadioactive qaste near beatty[END_REF][START_REF] Phillips | Mineralogy of selected geological deposits from the United Kingdom and the Republic of Ireland as possible capping material for low-level radioactive waste disposal facilities[END_REF][START_REF] Nasir | A simulator for modeling of porosity and permeability changes in near field sedimentary host rocks for nuclear waste under climate change influences[END_REF].

Fischer [START_REF] Fischer | Hydrogeologic factors in the selection of shallow land burial sites for the disposal of low-level radioactive waste[END_REF][START_REF] Fischer | Sediment properties and water movement through shallow unsaturated alluvium at an arid site for disposal of low-level eadioactive qaste near beatty[END_REF] undertook studies to determine the rates and directions of water movement through unsaturated sediments for the assessment of the contaminant movement near an arid LLW disposal facility. Phillips et al. [START_REF] Phillips | Mineralogy of selected geological deposits from the United Kingdom and the Republic of Ireland as possible capping material for low-level radioactive waste disposal facilities[END_REF] studied the mineral behavior and transformations brought on by climate change may affect the long-term performance of the cap and even the LLW disposal facility. Nasir et al. [START_REF] Nasir | A simulator for modeling of porosity and permeability changes in near field sedimentary host rocks for nuclear waste under climate change influences[END_REF] investigated that both permeability and porosity of the near field sedimentary host rocks for the LLW/ILW disposal increase as a result of climate change. In summary, the evaluation of waste covers in terms of waste isolation and contaminant transport requires details of subsurface flows which are significantly affected by climatic fluctuations. Specifically, the major threat to the integrity of nuclear waste isolation in the near-surface type of disposal facility is the water infiltration due to the potential movement of dissolved and mobilized radioactive constituents.

Therefore, considering the long life of radioactive waste, it is of paramount importance to well determine the hydro-geologic characteristics of an LLW/ILW disposal facility in order to limit water flow in the vicinity of the disposal facility [START_REF] Fischer | Hydrogeologic factors in the selection of shallow land burial sites for the disposal of low-level radioactive waste[END_REF][START_REF] Fischer | Sediment properties and water movement through shallow unsaturated alluvium at an arid site for disposal of low-level eadioactive qaste near beatty[END_REF].

The soil hydraulic condition in the overburden of LLW/ILW disposal facilities is governed by precipitation and evapotranspiration which are associated with the changing climate conditions. Generally, long-term climatic cycles present a low frequency and small amplitude, having less impact on the capping material. However, the low-frequency extreme weather events, such as a long-term drought, may result in significant damage to cap materials [START_REF] Phillips | Mineralogy of selected geological deposits from the United Kingdom and the Republic of Ireland as possible capping material for low-level radioactive waste disposal facilities[END_REF][START_REF] Smith | Natural physical and biological processes compromise the long-term integrity of compacted clay caps[END_REF]. The fundamentals of the safety assessment of waste disposal facilities of LLW/ILW/HLW in Belgium were discussed by van Geet et al. [START_REF] Van Geet | Climate evolution in the long-term safety assessment of surface and geological disposal facilities for radioactive waste in Belgium[END_REF], emphasizing the importance of considering climate evolution. However, to the authors' knowledge, no study has been undertaken to investigate the soil-atmosphere interaction in the LLW/ILW overburdens under the effect of future climate conditions.

This study aims at better understanding the soil-atmosphere interaction in the overburden of the LLW/ILW disposal facility, through identifying the soil coupled hydro-thermal responses to the future climate changes. A numerical approach combining a coupled hydro-thermal model and a soil-atmosphere interaction model was employed and implemented through the Finite Element Method using FreeFem++ code [START_REF] Hecht | FreeFem++, a tool to solve PDE's numerically[END_REF]. Considering climate analogues, actual meteorological data collected from Valencia El Saler station was employed as the future climate condition of the studied site located in middle France. Firstly, the meteorological data in the year 2000 collected at the time intervals of 30 min, daily, weekly, and monthly, were used to investigate the effect of the time scale of climate conditions on the hydro-thermal behavior of the overburden soil. Moreover, the influence depths in terms of soil water content and temperature at different time scales were examined, respectively. Secondly, the meteorological data at the time scale of 30 min for a longer period from 2000 to 2006 (7 years) was employed to investigate the long-term soil-atmosphere interaction and the influence depths in terms of soil temperature and water content.

Investigated site

The studied site which was devoted to the storage of short-lived LLW/ILW was located at Soulaines (Aube), France. Figure 1a plots its stratigraphic profile, including three layers: a layer of nature backfill soil, a 5 m superficial layer and a natural Teguline clay layer of 50 to 70 m thickness. Specifically, the excavated zone is at a depth of -20~-30 m and the storage space for LLW/ILW is at a depth of -25~-35 m.

For further study, the studied region is simplified as three layers with homogeneous soil for each (Figure 1b). The primary soil information of each layer is detailed in Table 1. The soil temperature at the bottom zone of the studied region was measured as 14°C [START_REF] Plas | Référentiel du site de la Communauté de Communes de Soulaines-Projet FAVL Communauté de Communes de Soulaines. Programme de reconnaissance[END_REF]. The local water table was close to the bottom boundary of the natural Teguline clay layer. The soil thermal conductivity, water retention curve and hydraulic conductivity curve of each layer are detailed as follows.

Layer 1 (L1)

In terms of silt clay as the backfill material of L1, its parameters used for the assumed simulations were estimated from results obtained on a soil comparable to that at the site. The variation of soil thermal conductivity versus its volumetric water content is expressed in a linear relationship by equation (1) [START_REF] Mickley | The thermal conductivity of moist soil[END_REF][START_REF] Pauly | Thermal conductivity of soils from the analysis of boring logs[END_REF] (Table 2) and plotted in Figure 2a. The soil water retention curve (Figure 2b) can be described by van Genuchten model [START_REF] Escario | Strength and deformation of partly saturated soils[END_REF][START_REF] Jahangir | A new coupled heat, moisture and air transfer model in unsaturated soil[END_REF][START_REF] Van Genuchten | Closed-Form Equation for Predicting the Hydraulic Conductivity of Unsaturated Soils[END_REF]. The relevant parameters in equation ( 2) are listed in Table 2. Based on the soil water retention curve, its hydraulic conductivity curve is determined by equation ( 3) and drawn in Figure 2c. The saturated soil hydraulic conductivity is estimated as 1.0× 10 -8 m/s [START_REF]Characteristic coefficients of soils n[END_REF].

Layer 2 (L2)

KD2 instrument [START_REF] Tang | A study on the thermal conductivity of compacted bentonites[END_REF] was used herein to measure the thermal conductivity of the compacted Teguline clay. A linear relationship between soil thermal conductivity and volumetric water content is considered to fit the measured results [START_REF] Vries | Thermal properties of soils[END_REF][START_REF] Cui | Field simulation of in situ water content and temperature changes due to ground-atmospheric interactions[END_REF]. The proposed relationship is expressed by equation ( 4) and presented in Figure 2a. Similar to the case of L1, van Genuchten model [START_REF] Van Genuchten | Closed-Form Equation for Predicting the Hydraulic Conductivity of Unsaturated Soils[END_REF] is used to describe the variations of soil water retention and hydraulic conductivity of L2. Based on the measured data obtained by Cuisinier and Masrouri [START_REF] Cuisinier | Détermination des propriétés hydromécaniques des argiles tégulines et des marnes de Brienne -Projet FAVL Rapport final[END_REF], the fitting curve is drawn in Figure 2b, providing the values of van Genuchten model parameters in Table 2. The hydraulic conductivity curve is determined with the measured saturated hydraulic conductivity 1.4×10 -9 m/s [START_REF] Cuisinier | Détermination des propriétés hydromécaniques des argiles tégulines et des marnes de Brienne -Projet FAVL Rapport final[END_REF] (equation (3) and Figure 2c).

Layer 3 (L3)

As far as the natural Teguline clay is concerned in L3, the thermal conductivity was measured by Zhang et al. [START_REF] Zhang | Anisotropic features of natural Teguline clay[END_REF], presenting slight variations versus soil dry density and water content. Therefore, its soil thermal conductivity is considered as a constant value of 2 W/(m.K) for the purpose of simplification. Based on the measured data obtained by Zeng et al. [START_REF] Zeng | Experimental study on swelling behaviour and microstructure changes of natural stiff teguline clays upon wetting[END_REF], the variations of soil water retention and hydraulic conductivity of L3 are described by equations ( 2) and (3). The fitting curves are plotted in Figures 2b and2c, respectively. The measured saturated hydraulic conductivity is 3.0×10 -12 m/s.

Meteorological information

Hallegatte et al. [START_REF] Hallegatte | Using climate analogues for assessing climate change economic impacts in urban areas[END_REF] presented a type of climate relocation based on the concept of spatial climate analogues, which specifies that a location that presently enjoys a climate close to the one another city will experience in the future (the end of the twenty-first century). Based on the acceptable analogues of climate relocation, the studied site in the central part of northern France may experience an eastern Spain type of climate in the future [START_REF] Kopf | Using maps of city analogues to display and interpret climate change scenarios and their uncertainty[END_REF][START_REF] Beck | Present and future köppen-geiger climate classification maps at 1-km resolution[END_REF]. Thereby, the actual meteorological data collected from Valencia El Saler station was employed to represent the future climate conditions that the studied site located in middle France may experience. It is worth noting that in the current knowledge of climate change, the predicted future local climate conditions are always attached with uncertainties due to the limitations of climate models, internal climate system processes and socio-economical, demographic or technological evolutions. The measured solar radiation, air temperature, latent heat, rainfall and the estimated actual evaporation rate from 01/01/2000 to 31/12/2000 are plotted in Figure 3 at different time scales (30 min, daily, weekly and monthly respectively). They were used to study the effect of climate conditions at different time scales on the interaction between soil and atmosphere. Figures 3a,3b, and 3c demonstrate the diurnal and seasonal variations of solar radiation, air temperature, and latent heat, with higher values in the summer period and lower values during the wintertime. However, it is observed that rainfall data does not vary seasonally (Figure 3d). Similar to the results of latent heat, evaporation data presents the highest value in summer (Figure 3e). In addition, the comparison of each climate factor at different time scales suggests that the smaller fluctuations are obtained at a longer time scale. Especially, the extreme climate conditions, such as the highest temperature and the most torrential rainfall events, can be identified at the time scale of 30 min. Furthermore, meteorological information at the time scale of 30 min in the period from 01/01/2000 to 31/12/2006 (7 years), was employed to study the long-term soil hydro-thermal behavior (Figure 4) in the future. The annual variations of solar radiation, air temperature, latent heat, rainfall, and the estimated evaporation are plotted in Figures 4a~e,respectively, displaying an apparent seasonality. The peak values of solar radiation (Figure 4a) and air temperature (Figure 4b) are observed in summertime and wintertime of each year. Specifically, the magnified image of solar radiation above 800 W/m 2 displays an increasing trend, reflecting the warming climate condition as time continues. The effect of climate change on the variations of latent heat and evaporation is identified by higher values of latent heat and evaporation in the wintertime of years

Numerical approach

Introduction of the numerical approach

A numerical approach combining a coupled hydro-thermal model and a soil-atmosphere interaction model developed by An et al. [START_REF] An | Numerical analysis of soil volumetric water content and temperature variations in an embankment due to soil-atmosphere interaction[END_REF] was employed in this study. The coupled hydro-thermal model was used to describe the coupled water and heat flow in unsaturated soil. The soil-atmosphere interaction model was applied to describe the continuous water and heat transfer between soil and atmosphere, which can be expressed by energy and mass balance, respectively. Details of the numerical formulation and computational aspects have been presented by An et al. [START_REF] An | Numerical analysis of soil volumetric water content and temperature variations in an embankment due to soil-atmosphere interaction[END_REF], and therefore, they are not repeated herein. On the first day of the year 2000, the soil volumetric water content was measured as 5.8% and the soil temperature was 11.67 ºC at the ground surface. Meantime, the soil temperature was measured to be 14 ºC at the saturated bottom boundary (BC1). Thereby, the initial conditions about soil temperature and water content/suction were defined in a linear relationship with depth. No water and heat transfer were considered at the lateral boundaries. Based on the soil-atmosphere interaction model, the soil heat flux and evaporation/infiltration were applied as the thermal and hydraulic conditions at the top boundary, respectively. The details are explained as follows:

Model dimensions and boundary conditions

Energy balance

In the case of heat transfer between soil and atmosphere, solar radiation is usually the only exterior heat resource. Specifically, the net solar radiation equals the sum of latent heat, soil heat, and sensible heat. The energy balance is described by:

n E R G L H = + + (5)
where Rn (W/m 2 ) is the net solar radiation flux; G (W/m 2 ) is the soil heat flux; LE (W/m 2 ) is the latent heat flux; H (W/m 2 ) is the sensible heat flux. The net solar radiation at 30 min is estimated by:

( ) ( ) ( ) 0.5 4 1 si n si c c e e d a so R R R a b a b e T R α σ     = - - + +         (6)
where the solar radiation Rsi (W/m 2 ) was measured as shown in Figure 3a; Ta (ºC) is the recorded air temperature (Figure 3b); the definition and values of other parameters were detailed by An et al. [START_REF] An | Numerical analysis of soil volumetric water content and temperature variations in an embankment due to soil-atmosphere interaction[END_REF].

The measured values of latent heat at different time scales are introduced in Figure 3c.

Considering the convection of airflow in soil-atmosphere interaction, the sensible heat was estimated following the same method presented by An et al. [START_REF] An | Numerical analysis of soil volumetric water content and temperature variations in an embankment due to soil-atmosphere interaction[END_REF]. With the obtained values of net solar radiation, latent heat and sensible heat fluxes at different time scales, the soil heat flux boundary condition at the soil surface at the corresponding scale can be determined based on equation [START_REF] Kim | A multi-objective optimization of initial conditions in a radioactive waste repository by numerical thermo-hydro-mechanical modeling[END_REF].

Mass balance

In terms of bare soil during rainfalls, water reaches the ground surface and infiltrates into the soil continuously until the rainfall rate exceeds the soil infiltration capacity. In that case, the process of runoff begins. However, it stops as soon as the rate of rainfall becomes lower than the actual rate of infiltration. Meanwhile, evaporation occurs spontaneously due to the gradient in temperature and relative humidity near the soil-atmosphere interface. The mass balance during soil-atmosphere interaction is expressed as:

nf off a P I R E = + + (7)
where P (m/s), Inf (m/s), Roff (m/s) and Ea (m/s) represent the rainfall, infiltration, runoff and actual evaporation rates on soil surface, respectively. Field rainfall was monitored at different time scales of 30 min, daily, weekly, and monthly (Figure 3d). Around 97% of the measured rainfall rates are less than 5.5 mm/30min, which means that most of the runoff rates are in the range from 0.025 to 0.05 mm/30min [START_REF] An | Assessment of Rainfall Runoff Based on the Field Measurements on an Embankment[END_REF]. Therefore, runoff is considered to be negligible in this study. Actual evaporation rate (Figure 3e) was estimated based on the values of latent heat:

( ) a E w v E L L ρ = (8)
where LE (W/m 2 ) is the latent heat flux (Figure 3c); Lv (J/kg) is the latent heat of water vaporization;

ρw represents the water density (kg/m 3 ). With the available information about rainfall and actual evaporation rates, the water flux boundary condition (Inf) at the soil surface can be estimated at different time scales.

Firstly, with the meteorological information collected every 30 minutes and soil parameters, the numerical investigation was performed at a time step of 30 min for the year 2000. Afterwards, the simulation scenarios at time scales of daily, weekly and monthly were implemented respectively, In terms of the soil temperature at the studied depths, they present a general increasing trend from the winter to the summer, approaching the peak values on 18/08/2000. Afterwards, they start to decline gradually until the end of the year 2000. The distinct seasonal variation of soil temperature is observed with larger values in the summer and lower values in the winter (Figure 6b), showing a good agreement with the heat-related climate factors (solar radiation, air temperature, etc.). Similar to the result of soil volumetric water content, the surface soil temperature shows more significant variations compared to other depths.

To further explore the influence depth of the soil-atmosphere interaction, the calculated soil volumetric water content and temperature profiles in the zone with depth 0~-21 m are presented in Figures 7a and7b, respectively. The soil volumetric water content below -4.8 m keeps nearly stable, indicating that the influence depth of climate conditions is limited to this value. Figure 7b shows that the influence depth of climate conditions is limited to 12 m depth in terms of soil temperature.

Furthermore, a larger amplitude is identified in the profiles of soil temperature, compared to that of soil volumetric water content, reflecting the significant contribution of the surface thermal boundary conditions. It is therefore necessary to investigate the influence depths of temperature and volumetric water content separately, as suggested by An et al. [START_REF] An | Numerical analysis of soil volumetric water content and temperature variations in an embankment due to soil-atmosphere interaction[END_REF].

Soil hydro-thermal behavior at different time scales

The variations of soil volumetric water content at different time scales (30 min, daily, weekly, and monthly) are plotted for depths 0 m (Figure 8a), -0.25 m (Figure 8b), -0.75 m (Figure 8c), and -1.5 m (Figure 8d), respectively. At each depth, a similar variation tendency is observed among the results of soil volumetric water content at different time scales. Generally, the results at the time scale of 30 min depict the maximum and minimum values, except the unexpected daily results on 21/03/2000. In the daily results on the soil surface (Figure 8a), soil water content increases sharply to 35% on 21/03/2000 and then exhibits a general decreasing trend with a few jumps due to rainfall events until 11/06/2000. The daily results show a consistent variation mode as the 30 min results but with a higher value over the dry periods, suggesting the continuous effect of the unexpected daily rainfall data on 21/03/2000. In the same rainfall event, a more significant increase in water content is observed in the 30 min results compared to other results. This suggests that a time scale as short as 30 min is capable of increasing the accuracy of simulation results by accelerating the iteration rate of soil-atmosphere interaction. Furthermore, the daily fluctuations of soil water content are more evident at the time scale of 30 min than others, especially for the period with intense rainfall (23/10/2000 to 31/12/2000). m). This is attributed to the effective mass interaction between soil and atmosphere at the time scale of 30 min. On one hand, rainfall events occur during some specific periods rather than continue at a constant rate for daily/weekly/monthly. Hence, soil water content varies more quickly due to the rainfall at the time scale of 30 min than those of other time scales. On the other hand, due to the monthly average evaporation rate at the soil surface, water content at the time scale of monthly decreases for a longer period compared to others (Figure 8d). Thereby, when the evaporation/rainfall at the time scale of 30 min is applied as the hydraulic boundary condition, they lead to a shallower influenced zone compared to the results of other time scales.

On the other hand, similar to that of 30 min results, the influence depths of climate conditions on soil temperature is limited to around 12.0~12.8 m at time scales of daily, weekly, and monthly.

The effect of time scale on the influence depth of climate conditions in case of soil temperature is not significant as the results of soil water content. It may be attributed to the participation of soil surface temperature in the estimation of sensible heat and hence the value of heat flux boundary condition, which may lead to the same amount of energy transfer between soil and atmosphere at different time scales. Additional work is required to further clarify this point. In the region below the influenced zone, both soil volumetric water content and temperature remain stable as their real initial conditions during the studied period. It is thereby suggested to measure the real initial soil conditions in the area of interest for the evaluation of LLW/ILW storage system's performance.

Conclusions

In this study, the effect of climate conditions on soil water content and temperature in the overburden of LLW/ILW disposal facility were numerically investigated at different time scales

1 m n r e s r S θ θ αψ θ θ - -   = = +   - (2)
( )

2 0.5 1/ e e S 1 1 S m m s K K   = --     (

  2003, 2004, 2005 and 2006 than those of the years 2000, 2001 and 2002. Besides, more frequent rainfall events (Figure 4d) are recorded in the years 2003, 2004, 2005 and 2006.

Figure 5

 5 Figure 5 displays the applied model dimensions: BC1 and BC 3 represent the bottom and top

  Figures 6a and 6b depict the variations of soil volumetric water content and temperature at depths

Figure 9

 9 Figure 9 plots the evolutions of the soil temperature at different depths at different time scales

4. 2 .

 2 Figure 11a plots the changes in soil volumetric water content at different depths (0 m, -0.25 m, -

  The influence depth of climate conditions on temperature increases as time continues. It is inferred to be related to the warming climate conditions produced by the increasing solar radiation from the year 2000 to the year 2006 in Figure7a.As shown in Figures12h and 13h, in the studied period of seven years (2000 to 2006), the influence depths of climate conditions are estimated to be larger than 20 m for both soil water content and temperature. It indicates that the overburden and the current storage space for LLW/ILW are potentially situated in the weather influenced zone in the future. Therefore, further investigation about the effect of soil water content and temperature on the performance of LLW/ILW storage system is required to minimize the negative weather influence on LLW/ILW storage and to provide a practical suggestion for future site selections. Based on the results of each year and seven years, it is inferred that the influence depths of climate conditions vary significantly in different studied periods. The differences are governed by the climate conditions of the studied periods. During the longer studied period (7 years), the influence of climate conditions on the variations of soil volumetric water content and temperature is accumulated, leading to a deeper influenced zone compared to that of each year. It may be attributed to the effect of wetting-drying cycles during the longer period on the changes of soil microstructure, allowing heat and water transport to a deeper zone.

(( 1 )( 2 )( 3 )( 4 )( 5 )

 12345 30 min, daily, weekly and monthly) and in the long term(7 years). Actual meteorological information data from Valencia El Saler station in Spain were used as the future climate conditions of the studied site in middle France based on climate analogues. The obtained results allow the following conclusions to be drawn: The variations of soil volumetric water content and temperature in the near-surface zone are mainly affected by the hydraulic (evaporation/infiltration) and thermal (soil heat flux) boundary conditions at the soil-atmosphere interface. At soil surface, distinct daily and seasonal variations of soil temperature can be observed, with higher values during the summer period and lower values in the wintertime. In the deeper zone, both soil volumetric water content and temperature show much smaller fluctuations compared to those of near soil surface zone. The calculated results in terms of soil volumetric water content and temperature present a similar variation tendency at the same depth for the four different time scales. However, in general, the results at the time scale of 30 min depict the maximum and minimum values because of the extreme climate events recorded at this time interval. The daily fluctuations of soil water content and temperature are more evident at the time scale of 30 min than those of other time scales, especially at soil surface point. As a result, the employment of meteorological information at a short time scale (30 min) can increase the accuracy of simulation results by capturing the extreme climate events and accelerating the iteration rate of soil-atmosphere interaction. Thereby, it is suggested to apply the meteorological information at a short time scale (30 min) to estimate the soil hydro-thermal behavior in the geotechnical and environmental contexts. In terms of influence depth for soil water content in the year 2000, it is limited to 4.8 m, 5.2 m, 11.6 m, and 11.6 at the time scale of 30 min, daily, weekly, and monthly, respectively.The mass transfer between soil and atmosphere at the time scale of 30 min is more effective than others. However, the influence depth in terms of soil temperature is limited to 12.0~12.8 m, indicating the insignificant effect of time scale. It may result from the same amount of energy transfer between soil and atmosphere at different time scales. Additional work is required to further clarify this point. The change of soil temperature in the long term (7 years) indicates that the peak temperatures of the summer periods show an increasing trend from 01/01/2000 to 31/12/2006, reflecting the effect of the warming climate on soil temperature. Moreover, the influence of climate conditions on soil hydro-thermal behavior is accumulated over time, leading to a deeper influenced zone (> 20 m) in a longer studied period. It indicates that the overburden and the current storage space for LLW/ILW are potentially situated in the weather influenced zone in the future. This study infers that a further investigation about the effect of soil water content and temperature variations on the performance of LLW/ILW storage system is required. It also enables a further detailed inspection of climate's role in the LLW/ILW storage system to minimize the negative influence of climate change on LLW/ILW storage and to provide a practical suggestion for site selections.

Figure captions Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .Figure 10 .Figure 11 .Figure 12 .Figure 13 .

 12345678910111213 Figure captions

FiguresFigure 1 .Figure 2 .

 12 Figures

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 4. Field meteorological information from 01/01/2000 to 31/12/2006 at the time scales of 30 min: (a) solar radiation, (b) air temperature, (c) latent heat, (d) rainfall and (e) actual evaporation rate (calculated)

Figure 7 .

 7 Figure 7. At the time scale of 30 min: (a) soil volumetric water content profiles and (b) temperature profiles at different times in zone 0~-20 m

Figure 12 .Figure 13 .

 1213 Figure 12. At the time scale of 30 min, soil volumetric water content profiles at different times in 728 zone 0~-20 m during different studied periods: (a) year 2001, (b) year 2002, (c) year 2003, (d) year 729 2004, (e) year 2005, (f) year 2006, and (g) from year 2000 to 2006 730

Table captions Table 1 .

 captions1 Soil properties of each layer in the study region

Table 2 .

 2 Soil thermal conductivity, water retention curve and hydraulic conductivity curve of each layer in the study region

Table 2 .

 2 Soil thermal conductivity, water retention curve and hydraulic conductivity curve of each layer in the study region

	Tables
		Table 1. Soil properties of each layer in the study region
	Layer	Soil	Depth (m)	Porosity	Dry density (Mg/m 3 )
	L1	Silty clay	0~-1	0.35	1.3
	L2	Compacted Teguline clay	-1~-36	0.37	1.7
	L3	Natural Teguline clay	-36~-71	0.256	2.0

Acknowledgments

The authors wish to acknowledge the support from ANDRA (Agence Nationale pour la Gestion des Déchets Radioactifs) and Ecole des Ponts ParisTech (ENPC). The authors would also like to express their gratitude to Mr. Arnaud Carrara in Fundación Centro de Estudios Ambientales del Mediterráneo (CEAM) for collecting and providing the meteorological data at Valencia El Saler station in Spain.