Andréa Geoffroy

Karima Habbas

Boglarka Zambo

Laetitia Schramm

Arnaud Duchon

Eric Flatter

Laetitia Fouillen

Julie Zumsteg

Dimitri Heintz

Jean-Louis Mandel

Yann Hérault

Hervé Moine
email: moine@igbmc.fr

Pioglitazone improves deficits of Fmr1-KO mouse model of Fragile X syndrome by interfering with excessive diacylglycerol signaling

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fragile X syndrome (FXS) is the leading cause of familial intellectual disability (affecting approximately 1 in 5000 males and 1 in 8000 females) and is associated with cognitive and behavioral deficits that can include anxiety, hyperactivity, hypersensitivity, stereotypies, memory deficits, sleeping problems [START_REF] Hagerman | Fragile X syndrome[END_REF]. FXS is due to the transcriptional silencing of the FMR1 gene encoding the fragile X mental retardation protein (FMRP) [START_REF] Nelson | The unstable repeats--three evolving faces of neurological disease[END_REF] or, more rarely, to mutations in FMRP itself [START_REF] De Boulle | A point mutation in the FMR-1 gene associated with fragile X mental retardation[END_REF][START_REF] Myrick | Fragile X syndrome due to a missense mutation[END_REF][START_REF] Quartier | Intragenic FMR1 disease-causing variants: a significant mutational mechanism leading to Fragile-X syndrome[END_REF]. FMRP is an RNA binding protein associated with numerous mRNA species in neurons [START_REF] Maurin | HITS-CLIP in various brain areas reveals new targets and new modalities of RNA binding by fragile X mental retardation protein[END_REF][START_REF] Darnell | FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism[END_REF][START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF][START_REF] Li | Identification of FMR1-regulated molecular networks in human neurodevelopment[END_REF][START_REF] Sawicka | FMRP has a cell-type-specific role in CA1 pyramidal neurons to regulate autism-related transcripts and circadian memory[END_REF] and in other cell types [START_REF] Ascano | FMRP targets distinct mRNA sequence elements to regulate protein expression[END_REF]. FMRP has been proposed to control the translation of a number of its mRNA targets in neurons and the alteration of neuronal physiology associated with FXS is linked to an excessive protein translation in (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09. [START_REF] Busquets-Garcia | Targeting the endocannabinoid system in the treatment of fragile X syndrome[END_REF].301762 doi: bioRxiv preprint 3 neurons resulting from the loss of FMRP control [START_REF] Darnell | The translation of translational control by FMRP: therapeutic targets for FXS[END_REF]. A protein synthesis-dependent synaptic plasticity, group 1 metabotropic glutamate receptor long term depression (mGluR1-LTD), is altered in various areas of the brain [START_REF] Huber | Altered synaptic plasticity in a mouse model of fragile X mental retardation[END_REF][START_REF] Koekkoek | Deletion of FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar eyelid conditioning in Fragile X syndrome[END_REF][START_REF] Rio | Disrupted inhibitory plasticity and homeostasis in Fragile X syndrome[END_REF] of Fmr1-KO mouse. Accordingly, several downstream effectors of the mGluR1-dependent signaling have been shown to be overactivated (e.g. PKC [START_REF] Deng | Increased Persistent Sodium Current Causes Neuronal Hyperexcitability in the Entorhinal Cortex of Fmr1 Knockout Mice[END_REF], Ras/MEK/ERK/Mnk [START_REF] Osterweil | Hypersensitivity to mGluR5 and ERK1/2 leads to excessive protein synthesis in the hippocampus of a mouse model of fragile X syndrome[END_REF][START_REF] Sawicka | Elevated ERK/p90 ribosomal S6 kinase activity underlies audiogenic seizure susceptibility in fragile X mice[END_REF], PIK3K/AKT [START_REF] Sharma | Dysregulation of mTOR signaling in fragile X syndrome[END_REF][START_REF] Gross | Excess phosphoinositide 3-kinase subunit synthesis and activity as a novel therapeutic target in fragile X syndrome[END_REF][START_REF] Gross | Increased expression of the PI3K enhancer PIKE mediates deficits in synaptic plasticity and behavior in fragile X syndrome[END_REF], CB1R [START_REF] Busquets-Garcia | Targeting the endocannabinoid system in the treatment of fragile X syndrome[END_REF][START_REF] Zhang | Enhanced endocannabinoid signaling elevates neuronal excitability in fragile X syndrome[END_REF][START_REF] Maccarrone | Abnormal mGlu 5 receptor/endocannabinoid coupling in mice lacking FMRP and BC1 RNA[END_REF], while others are defective (Rac/PAK [START_REF] Chen | Physiological activation of synaptic Rac>PAK (p-21 activated kinase) signaling is defective in a mouse model of fragile X syndrome[END_REF]). In agreement with these data, the phosphorylation of eIF4E by Mnk [START_REF] Gkogkas | Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes[END_REF] and of S6 by ERK [START_REF] Sawicka | Elevated ERK/p90 ribosomal S6 kinase activity underlies audiogenic seizure susceptibility in fragile X mice[END_REF] is increased and an excess of protein synthesis is observed in vitro and in vivo notably in hippocampal and cortical neurons of Fmr1-KO mouse [START_REF] Qin | Postadolescent changes in regional cerebral protein synthesis: an in vivo study in the FMR1 null mouse[END_REF][START_REF] Dolen | Correction of fragile X syndrome in mice[END_REF]. How exactly these alterations result from the loss of FMRP remains unclear.

We previously identified that diacylglycerol kinase kappa mRNA (DGKk) is the mRNA species most bound by FMRP in dissociated cortical neurons from new born mice [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF]. In addition, DGKk expression is severely reduced in brain of Fmr1-KO mouse and the lack of FMRP leads to a decreased DGK activity in the neurons of Fmr1-KO mouse and in the brain of FXS patients causing a perturbation of diacylglycerol/phosphatidic acid homeostasis [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF].

Moreover, the knockdown of DGKk in the brain of wild-type mouse recapitulates main Fmr1-KO behaviors, and DGKk reexpression in Fmr1-KO hippocampal slices rescues the abnormal dendritic spines morphology [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF]. These data support a model where DGKk dysregulation plays a pivotal role in FXS pathomechanism [START_REF] Tabet | Fragile X syndrome: Are signaling lipids the missing culprits?[END_REF].

A prediction of this model of FXS states that interfering with DGKk dysregulation could be beneficial for FXS condition. Pioglitazone (PGZ), a thiazolidinedione drug approved for type 2 diabetes treatment was shown to activate DGK activity and inhibit the DAG-PKC signaling [START_REF] Verrier | PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase[END_REF]. Thus, PGZ could have a potential to correct some of the defects associated with DGKk loss of activity. In this study, we tested the impact of PGZ in neurons in vitro and on the Fmr1-KO mouse to rescue the DAG-PKC signaling-related defects of FXS. We first confirmed that DAG-PKC signaling is excessive in Fmr1-KO neurons and PGZ normalized (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09. [START_REF] Busquets-Garcia | Targeting the endocannabinoid system in the treatment of fragile X syndrome[END_REF].301762 doi: bioRxiv preprint 4 these alterations. Then, after defining that PGZ is efficiently delivered to brain by intraperitoneal (IP) administration route, we showed that PGZ treatment improved several of Fmr1-KO main behavioral defects, emphasizing the importance of DGKk dysregulation in FXS and highlighting a possible new intervention mean.

Materials and methods

Ethics statement

Animal work involved in this study was conducted according to relevant national comité national de réflexion éthique en expérimentation animale with approval APAFIS#5874-20l6062915583967 v2 and international guidelines (86/609/CEE).

Animal housing

In vivo experiments were conducted in Fmr1-KO2 male mice (E.J. [START_REF] Mientjes | The generation of a conditional Fmr1 knock out mouse model to study Fmrp function in vivo[END_REF]. C57BL/6J Fmr1 +/y males (Janvier Labs, France) were crossed with C57BL/6J Fmr1 +/females. At weaning age (4 weeks), male mice were grouped by 3 or 4 individuals from same age and genotype in individually ventilated cages (GM500, Tecniplast, UK), with poplar shaving bedding (Lignocell Select, JRS, Germany), and maintained under standard conditions, on a 12-h light/dark cycle (7h/19h), with standard diet food (standard diet D04, Scientific Animal Food and Engineering, France) and water available ad libitum. Mice in the same cage received the same treatment and were transferred to the phenotyping area the following week.

Animal treatment

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 5 Pioglitazone (E6910, Sigma-Aldrich) was administered at 20 mg/kg/day using several administration routes. Food pellets containing vehicle or PGZ (130mg PGZ/KG D04, Safe diet, Augy, France, based on 4-5 g/day pellet consumption) were given from weaning for three weeks before starting the behavioral experiments and continued during the tests.

Subcutaneous implants for 60 days treatment (Innovative Research of America, Sarasota, FL, Cat. No. SX-999) prepared with 30 mg PGZ per pellet or vehicle (based on 60 x 0,5mg/day delivery) were placed under dorsal neck skin by sterile surgery. Intraperitoneal injections of PGZ solution at 3 mg/ml in NaCl 0.9%, DMSO 30% (v/v) (or vehicle) were performed daily starting at 5-week of age for one week prior to starting the behavioral tests and continued during the tests (with injection at end of each day test). Well-being of animals was controlled by daily visual control and weekly weighing.

Cortical neuron cultures

Cortical neuron cultures were prepared as in [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF]. Briefly, cortices of WT or Fmr1-KO (Fmr1 - /y ) E17.5 embryos were dissected in PBS 1X, 2.56 mg/mL D-glucose, 3 mg/mL BSA and 1.16 mM MgSO 4 , incubated for 20 min with 0.25 mg/mL trypsin and 0.08 mg/mL DNase I and mechanically dissociated after supplementation of medium with 0.5 mg/mL trypsin soybean inhibitor, 0.08 mg of DNase I and 1.5 mM MgSO 4 . Neurons were seeded on poly-L-lysine hydrobromide-coated six-well culture plates in Neurobasal Medium (NBM, GIBCO) supplemented with B27, penicillin/streptomycin and 0.5 μM L-glutamine. Primary cortical neurons were used for experiments after 8 days of culture at 37°C, 5 % CO 2 . Where indicated, cultures were treated with addition of pioglitazone solution and/or puromycin at the indicated concentrations and times. After treatment, cells were immediately washed with ice-cold PBS and lysed in 4X Laemmli buffer.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 6

Pioglitazone measurement in brain

Mouse brains (200 mg frozen ground homogenate) were homogenized mechanically with 1:1 w/v H 2 O and then diluted at 1/3 v/v in H2O. Rosiglitazone (Sigma, 2.5ng solubilized in 5µL DMSO/H2O 1:1, v/v) was added as internal standard and 500µl methanol was added to 95µl of brain homogenate. Samples were vortexed for 5min at maximum speed and centrifuged for 20min at 14.000 g at 4°C. 300µL of supernatant (equivalent to 15µg tissues) were transferred to new collecting tube. Samples for establishing standard curve of PGZ were prepared in same conditions by adding 1000ng, 500ng, 250ng, 125ng, 62.5ng or 31.25ng PGZ in (Sigma).

Rosiglitazone and PGZ were identified and quantified by UPLC-MS/MS. Quantitative profiles were analyzed using an EVOQ Elite LC-TQ (Bruker Daltonics) equipped with an electrospray ionization source and coupled to an HTC Pal-xt (Bruker Daltonics) and an Advance UHPLC system (Bruker Daltonics). Chromatographic separation was achieved using an Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm; Waters) and pre-column. The mobile phase consisted of (A) water and (B) methanol, both containing 0.1% formic acid. The run started by 2 min of 95 % A, then a linear gradient was applied to reach 99 % B at 10 min, followed by isocratic run during 1,5 min. Return to initial conditions was achieved in 1 min and equilibrate for 2,5 min, with a total run time of 15 min. The column was operated at 40°C with a flow rate of 0.35 ml/min, injecting 5 μL samples. Nitrogen was used as the drying and nebulizing gas. The nebulizer gas flow was set to 35 L/h, and the desolvation gas flow to 30 L/h. The interface temperature was set to 350°C and the source temperature to 300°C. The capillary voltage was set to 3.5 kV; the ionization was in positive mode. Low-mass and highmass resolution was 2 for the first mass analyzer and 2 for the second. Data

DAG measurements

Mouse brains (100 mg frozen ground homogenate) were homogenized with 1 ml H 2 0 and extracted by vortexing with 2 ml of chloroform/methanol 2:1 (v/v) and 10 μl of synthetic internal standard (DAG 15:0/15:0, Sigma Aldrich), sonicated for 30 s, vortexed, and centrifuged. The lower organic phase was transferred to a new tube, and the upper aqueous phase was reextracted with 2 ml of chloroform. Organic phases were combined and evaporated to dryness under nitrogen. Lipid extracts were resuspended in 50 μL of eluent A, and a synthetic internal lipid standard (DAG 17:0/17:0, Nu-Check Prep) was added.

LC-MS/MS (MRM mode) analyses were performed with mass spectrometer model QTRAP 5500 (ABSciex) coupled to a LC system (Ultimate 3000; Dionex). Analyses were achieved in positive mode. Nitrogen was used as curtain gas (set to 20), gas1 (set to 25), and gas2 (set to 0). Needle voltage was at +5,500 V without needle heating; the declustering potential was set at +86 V. The collision gas was also nitrogen; collision energy was adjusted to +34 V. Dwell time was set to 3 ms. Reversed-phase separation was carried out at 30 °C on a Phenomenex Luna 3u C8 column (150 mm × 1 mm, 3-μm particle size, 100-Å pore size). Eluent

Immunoblot analyses

Immunoblot analyses were performed on 14 days-old E19 cortical primary neurons lysed in 4X Laemmli buffer (equivalent to 15 µg total proteins) or brain homogenate (30mg of frozen ground total brain) solubilized in RIPA lysis buffer (140 mM NaCl, 0.5% sodium deoxycholate (w/v), 1% Nonidet P-40 (v/v), 0.1% SDS (w/v), and protease inhibitors (Roche Applied Science), lysed by three cycles of freezing/thawing, centrifuged at 4°C for 30 min at 15,000 g. Brain extracts (equivalent to 15 µg total proteins) were mixed with an equal volume of 2X Laemmli buffer. Immunoblotting were performed as described previously [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF]. Proteins were denatured by heating the mixture for 5 min at 95°C, and then resolved by 10% SDS-PAGE. Separated proteins were transferred using a Mini Trans-Blot (Biorad) cell onto PVDF Immobilon P membrane (Millipore). Membranes were blocked for one hour with TBS-T 1X (Tris-Buffer Saline, pH 7.4 and 0.1% Tween-20 v/v) containing 5% BSA (w/v) or 5% nonfat dry milk. Membranes were incubated overnight at 4°C with primary antibodies diluted in TBS-T buffer containing 5% w/v BSA or milk as follows: anti-FMRP (1C3, 1/10,000, in house generated antibody) anti-p-EIF4e (Ser209, 1/1000 BSA, #9741, Cell Signaling), anti-EIF4 (1/1000 BSA, #9742, Cell Signaling), anti-p-PKC pan (βII Ser660, Rb, 1/1000 BSA #9371, Cell Signaling), anti-PKCα (1/2000 BSA, sc-208, Santa Cruz), anti-puromycin (12D10, 1/2000, Sigma-Aldrich), anti-S6 (5G10, 1/1000 #2217S, Cell Signaling), anti-p-S6 (Ser235/236, 1/1000, 2211S Cell Signaling), anti-GAPDH (MAB374, 1/10000, Merck) was used as an internal standard. Membranes were washed in TBS-T buffer and then incubated for an hour at room temperature with the corresponding horseradish peroxidase-conjugated preadsorbed secondary antibody (1/2000, blocking solution corresponding, Molecular Probes).

Membranes were washed in TBS-T buffer. Immunoreactive bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, cat. WBKLS0100).

Immunoblot pictures were acquired using LAS600 GE Amersham and density of the resulting (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 9 bands was quantified using ImageJ and statistical significance assessed using repeated measures analysis of variance (ANOVA) with Fisher's post hoc comparisons (GraphPad Prism 7 software, La Jolla, CA, USA).

RT-qPCR

Total RNAs were extracted with standard Trizol protocol (Trizol, Ambion) from 14 days-old E19 cortical primary neurons (equivalent to one well of 6-well plate) or ground brains (30mg). Total RNA (0,5μg) was treated with DNase (TURBO DNA-free™ Kit, Ambion) following manufacturer instructions. First strand cDNA was synthesized with SuperScript II Reverse Transcriptase kit (Invitrogen) following manufacturer instructions used with random primer mix. qPCR was performed on cDNA dilutions of 1/20 in the presence of 7.5-pmol primers with QuantiTect SYBR Green PCR Master Mix (Qiagen) on a Lightcycler 480 (Roche), using specific primers (Table S1). Relative quantifications were performed with Ribosomal Protein Lateral Stalk Subunit P0 (Rplp0) and Actb as internal standards. Results were expressed as arbitrary units by calculating the ratio of crossing points of amplification curves for cDNAs and internal standard by using the LightCycler 480 software (version LCS480 1.5.0.39, Roche Diagnostics). Fold change between groups were determined by using the following 2e-∆∆ct method. Error rates were calculated with square root ((SD control/SD treated) 2 + (fold change) 2 ).

Behavior analyses

One week after starting of PGZ treatment behavior tests were conducted to evaluate locomotor activity and anxiety, cognition, autistic-like behaviors and sensori-motor gating.

Animals were transferred to the experimental room 30 min before each experimental test. The tests were performed in the following order: open field, novel object recognition, rotarod, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 10 stereotypy, sociability, actimetry, pre-pulse inhibition, audiogenic seizures (Fig. S1). After each test, material was cleaned with water, 50% ethanol, water, and then dried with paper towels to minimize olfactory cues. IP treatment was applied after behavioral sessions to minimize impact of injection with tests. Behaviors were either analyzed automatically or by an observer blind to the conditions.

Open field

Locomotor activity and anxiety of animals were evaluated in an open field test. Animals were allowed to freely explore the new environment of plexiglass square arena (44 x 44cm x 16 cm, Panlab Harvard apparatus IR ACTIMETER, Bioseb) at a luminosity of 150 +/-15 lux.

Activity was recorded for 30 min using ActiTrack software (version 2.55). Infra-red lasers at bottom and top of arena recorded vertical and horizontal activity. For the analysis, the arena was divided between a 10-cm wide peripheral zone and a central 30cm x 30cm zone to evaluate locomotion, exploration, hyperactivity and anxiety. Distance moved and time spent in center or periphery zones, velocity, speeds and rearing were scored. Parameters were quantified in cumulative (0-30min) and windows (0-10min, 10-20min and 20-30min).

Novel Object recognition

Learning and memory abilities of mice were evaluated using the novel object recognition test.

Rotarod

Locomotors coordination was evaluated during 4 successive session-tests performed at a luminosity of 115 +/-15 lux on a rotarod system composed of a rotating plastic bar covered by grey rubber foam (5 cm in diameter, Panlab Harvard apparatus LE 8200, Bioseb). The animals were first habituated to stay on the rod for 30 s at a constant speed of 4 rotations per minute (rpm), facing to the direction of rotation. The test was performed on the rotating rod with acceleration steps from 4, 10, 16, 22, 28, 34 to 40 rpm over 5 minutes. First session was considered as a training session, followed by 3 trial sessions separated by 10 min resting. The test was stopped when the mouse fell or when there was more than one passive rotation. The latency to fall and the maximal speed before falling were recorded.

Stereotypy

Stereotypy was evaluated on animals isolated in a new home cage (GM 500 techniplast, floor area 501 cm² at a luminosity of 70 +/-5 lux) with sawdust bedding for 10min and measuring

Actimetry

Locomotor and circadian activities were measured in an actimetry recording systems coupled to BMB software (Imetronic). Mice were placed in activity boxes (25 x 20 x 15 cm). Infra-red lasers at the bottom and the top of the square inform about vertical and horizontal activity when cut by mouse body (15 mm for horizontal activity and 30 mm for vertical activity).

Activity was recorded during 24 h, starting at 06:00 pm (1h before light switch). Rearing events, front-and back-yard activities, round trips, food and water consumption were recorded.

Pre-Pulse inhibition (PPI)

PPI was measured using acoustic startle test with SR-LAB apparatus and software (San Diego Instruments, USA). Mice were placed individually in the PPI test plexiglass tube (12,7cm (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 13 long, 3,8cm internal diameter, 5,0cm external diameter) of the startle box (constant luminosity of 70 +/-5 lux and white noise background). Habituation phase was performed for 5 min.

Then mice were exposed to pulses of white sound of 20 msec duration and varying intensity: +6, +12, +18, and +24 dB over background levels (namely 72, 78, 84, and 90 dB). Each intensity was applied eight times, in a randomized order with variable intervals (10-20 sec) between the onset of each pulse. A total of 130 readings of the whole-body startle response were taken at 0.5-msec intervals (i.e., spanning across 65 msec), starting at the onset of the pulse stimulus. The average amplitude (in arbitrary units) over the 65 msec was used to determine the stimulus reactivity and further averaged across trials.

Statistical analyses

Data were analyzed using an analysis of variance (ANOVA) with Fisher's post hoc comparisons. ANOVA with repeated measures analysis were carried out when required by the experimental plan to assess complementary statistical effects. In some designs, statistical analysis was performed using Student's t-tests. For all analysis p-value p<0.05 was considered to indicate significance. All analyses were performed using Statview 5 software for Windows (SAS Institute, Berkley, CA, USA) or GraphPad Prism (GraphPad Software, La Jolla, CA, USA) software.

Results

DAG signaling is excessive in Fmr1-KO neurons and is normalized by DGK-agonist pioglitazone (PGZ)

We previously showed that the level of second messenger diacylglycerol (DAG) is increased in Fmr1-KO cortical neurons and in cerebellum of FXS patient, in agreement with the observed loss of DGKk synthesis in FXS condition [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF]. DGKk is one of the ten DGK (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 14 enzymes that control DAG homeostasis [START_REF] Merida | Diacylglycerol kinase control of protein kinase C[END_REF]. Among the first direct effectors of DAG are protein kinase C enzymes (PKC), which are activated through sequential phosphorylation steps [START_REF] Merida | Diacylglycerol kinase control of protein kinase C[END_REF], notably at carboxy-terminal hydrophobic site Ser660 [START_REF] Keranen | Protein kinase C is regulated in vivo by three functionally distinct phosphorylations[END_REF]. In agreement with an increase of DAG-induced PKC activation, we measured an elevation of PKC phosphorylation in cultured cortical neurons from Fmr1-KO mice compared to their wild-type (WT) littermate control cultures (Fig. 1A). As a possible mean to interfere with or compensate for the loss of DGKk in Fmr1-KO cortical neurons, we tested the impact of DGK-agonist pioglitazone (PGZ), a molecule with PPARg agonist activity reported to inhibits DAG-PKC signaling pathway by activating DGK activity [START_REF] Verrier | PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase[END_REF]. Remarkably, the treatment of cortical neuron cultures with PGZ at 10 µM for 1h normalized the phosphorylation of PKC in Fmr1-KO while its level in WT remained unaffected by the treatment (Fig. 1B). Several protein effectors of ERK pathway have been reported to be over-activated in neocortex of Fmr1-KO mouse [START_REF] Sawicka | Elevated ERK/p90 ribosomal S6 kinase activity underlies audiogenic seizure susceptibility in fragile X mice[END_REF] and in FXS patients [START_REF] Wang | Activation of the extracellular signal-regulated kinase pathway contributes to the behavioral deficit of fragile xsyndrome[END_REF]. Consistent with an increased ERK activity, eukaryotic initiation factor 4E (eIF4E) phosphorylation is elevated in the brain of individuals with FXS and Fmr1-KO mice [START_REF] Gkogkas | Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes[END_REF]. Treatment of cortical neuron cultures with PGZ normalized phosphorylation of eIF4E in Fmr1-KO neurons while the WT level was non-significantly affected by the treatment (Fig. 1C).

As a possible consequence of increased DAG signaling and eIF4E activation, Fmr1-KO mice exhibit elevated basal levels of protein synthesis rate in brain [START_REF] Bhattacharya | Genetic removal of p70 S6 kinase 1 corrects molecular, synaptic, and behavioral phenotypes in fragile X syndrome mice[END_REF]. The 10-20% increase of protein synthesis rate previously reported in hippocampal slices [START_REF] Qin | Postadolescent changes in regional cerebral protein synthesis: an in vivo study in the FMR1 null mouse[END_REF][START_REF] Dolen | Correction of fragile X syndrome in mice[END_REF] could not be confirmed in cortical dissociated neurons cultures, possibly because the variability between neuron cultures was in the same range of effect. We did however observe a reduction of the translation rate in both Fmr1-KO and WT neurons after PGZ treatment (Fig. 1D), with a more pronounced dose reduction in WT compared to Fmr1-KO, possibly linked to a higher basal translation rate in Fmr1-KO, confirming the ability of PGZ to reduce protein synthesis rate.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 15 Not only ERK but also mTOR signaling pathways is hyper-activated in Fmr1-KO mice [START_REF] Gkogkas | Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes[END_REF][START_REF] Bhattacharya | Genetic removal of p70 S6 kinase 1 corrects molecular, synaptic, and behavioral phenotypes in fragile X syndrome mice[END_REF][START_REF] Osterweil | Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome[END_REF]. Consistent with mTOR signaling activation, protein S6 phosphorylation is increased in Fmr1-KO neurons and this increase is normalized with PGZ treatment (Fig. 1E).

Pioglitazone is delivered efficiently in brain of Fmr1-KO mouse by intraperitoneal injection

PGZ has been demonstrated to cross blood brain barrier [START_REF] Grommes | The PPARgamma agonist pioglitazone crosses the blood-brain barrier and reduces tumor growth in a human xenograft model[END_REF] and, based on our above data, appeared as an interesting drug to test on Fmr1-KO mouse FXS model. To define the best route to deliver PGZ efficiently in brain, we compared several modes of administration: subcutaneous diffusing micropellet, food (solid pellets), daily intraperitoneal injection (IP).

We chose a 20 mg/kg mouse/day dose as an average upper high dose in mouse based on literature survey. Treatment was started on 5-week-old mice and continued for three weeks.

PGZ quantification in brain by HPLC-MS/MS revealed that IP administration enables to reach the highest dose of PGZ in the brain (19.4±9 nM) compared to food (3.7±0.9 nM) and subcutaneous administration (0.42±0.26nM), with no significant variation between Fmr1-KO and WT (Fig. 2). Based on these data we chose to administer PGZ via IP route.

Pioglitazone daily IP treatment ameliorates Fmr1-KO behaviors, macro-orchidism and DAG

Five-week-old Fmr1-KO C57BL/6J hemizigous males and their wild-type (WT) littermates were injected IP with PGZ (20 mg per kg bodyweight per day) or vehicle for 7 days before starting the behavioral tests (Fig. S1) and IP injections were continued daily after each behavioral session (i.e. 19 d total). In the open field test, vehicle-treated Fmr1-KO mice did not show specific alterations compared to vehicle-treated WT mice in all the parameters analyzed (distance travelled, % time in center in arena, distance travelled in center of arena, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 16 number of rearings (Fig. S2A-C), suggesting that, in the conditions where the animals were handled, the influence of the genotype was not visible on the locomotor activity and anxiety of the animals. Noticeably, both WT and Fmr1-KO PGZ-treated mice had an average reduction of distance travelled and rearing numbers by approximately 20%, appearing as an unexpected previously unreported effect of the drug on mouse locomotor and rearing activity.

In the novel object recognition test, vehicle-treated Fmr1-KO mice showed a clear alteration of their ability to discriminate a novel vs a familiar object (recognition index = 49.89±1.33, not different from chance = 50%, Fig. 3A) compared to vehicle-treated WT (recognition index = 66.95±2.38). This difference was not the consequence of an altered exploration time of objects since this parameter was similar in both genotype and treatment groups (Fig. S3A).

PGZ-treated Fmr1-KO mice had a fully rescued ability to discriminate the novel object (recognition index =68.33±2.28 not significantly different from WT 66.95±2.38). Treatment had no visible effect on the WT mice discrimination and did not modify the exploration parameters of both genotypes.

In the free sociability dyadic reciprocal social interaction test, vehicle-treated Fmr1-KO mice showed an increased number of contacts with shorter contact duration time compared to WT mice (Fig. 3B,C). In contrast, PGZ-treated Fmr1-KO mice were not different from vehicletreated WT, suggesting that treatment had normalized their social behavior. In contrast, vehicle-treated WT mice were not visibly behaving differently from their PGZ-treated WT littermates in this test.

Vehicle-treated Fmr1-KO mice showed a significant increase of number of digging compared to vehicle-treated WT (Fig. 3D) while PGZ-treated Fmr1-KO mice were not different from vehicle-treated WT suggesting also that treatment had normalized their digging behavior. PGZ-treated WT showed a trend to have a decreased number of digging compared to vehicle-(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint treated WT but this was not significant. Vehicle-treated Fmr1-KO mice showed a significant decrease of grooming duration compared to vehicle-treated WT (Fig. 3E) while PGZ-treated Fmr1-KO mice were not different from vehicle-treated WT suggesting also that treatment had normalized their behavior. PGZ-treated WT showed no difference compared to vehicletreated WT.

In the rotarod test, no influence of the genotype or of the treatment was observed, neither in habituation nor in the following sessions (Fig. S3B). Prepulse inhibition test was performed but there was no significant difference between genotypes and the treatment groups (Fig. S3C), but this test showed variability across the studies [START_REF] Frankland | Sensorimotor gating abnormalities in young males with fragile X syndrome and Fmr1knockout mice[END_REF][START_REF] De Vrij | Rescue of behavioral phenotype and neuronal protrusion morphology in Fmr1 KO mice[END_REF][START_REF] Spencer | Exaggerated behavioral phenotypes in Fmr1/Fxr2 double knockout mice reveal a functional genetic interaction between Fragile X-related proteins[END_REF], possibly related to the sensitivity of recording methods used.

Macro-orchidism is a hallmark of post-adolescent male individuals with FXS (1) and is present in Fmr1-KO mice [START_REF] Mientjes | The generation of a conditional Fmr1 knock out mouse model to study Fmrp function in vivo[END_REF]. PGZ administration for 19 d reduced the testicular weight in Fmr1-KO mice to the same level as the WT treated mice (Fig. 3F). Noticeably, PGZ had a trend to reduce the testicular weight in WT mice, suggesting a possible impact of PGZ both in normal and pathological context. Meanwhile, no significant difference for total or brain weight was observed between genotype and treatment groups (Fig. S4A,B). PGZ treated animals showed a trend to have a reduced weight gain during the time of tests, but this was not statistically significant. Noticeably, while the body composition of vehicle-treated Fmr1-KO mice was not significantly different from vehicle-treated WT mice, PGZ treatment induced a significant increase of water retention in the tissues of Fmr1-KO mice (Fig. S4C-G), a well-known side effect of PGZ treatment [START_REF] Niemeyer | Thiazolidinedione-induced edema[END_REF]. Diacylglycerol (DAG) is increased in Fmr1-KO cortical neurons and in cerebellum of FXS patient, in agreement with a loss of DGKk activity in FXS neurons [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF]. The level of several main DAG species was found to be increased in vehicle-treated Fmr1-KO total brain extracts, notably main DAG species 38:4, and their level was normalized in PGZ-treated Fmr1-KO mice (Fig. 4).

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Discussion

In this study, we tested the ability of diacylglycerol kinase agonist pioglitazone (PGZ) to correct the symptoms of Fmr1-KO mouse model of fragile X syndrome. The rationale of this study is based on the fact that diacylglycerol kinase kappa (DGKk), a master regulator whose mRNA is a main target of FMRP in neurons, is under-expressed in the brain of Fmr1-KO mouse and signs of reduction of DGK activity (namely DAG/PA imbalance) are observed both in Fmr1-KO mouse and in FXS patients [START_REF] Tabet | Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons[END_REF]. Substrate of DGKk, DAG, is a prominent modulator of synaptic transmission probably in all types of synapses [START_REF] Kim | Diacylglycerol kinases in the regulation of dendritic spines[END_REF][START_REF] De Jong | Presynaptic signal transduction pathways that modulate synaptic transmission[END_REF][START_REF] Almena | Shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling[END_REF] that is produced by phospholipase C (PLC) from abundant phosphatidylinositol 4,5-bisphosphate (PIP2) membrane stores [START_REF] Kadamur | Mammalian phospholipase C[END_REF] after stimulation of G protein-coupled receptors such as mGluRI. The excess of DAG originating from the loss of DGKk activity could play a prominent role in FXS pathomechanism because DAG, through activation of its main effector protein kinase C (PKC) [START_REF] Keranen | Protein kinase C is regulated in vivo by three functionally distinct phosphorylations[END_REF], could be responsible of mitogen activated protein kinase (MAPK/ERK) [START_REF] Schonwasser | Activation of the mitogenactivated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes[END_REF] or Ras/phosphatidylinositol-3-kinase (PI3K)/AKT pathways [START_REF] Castellano | RAS Interaction with PI3K: More Than Just Another Effector Pathway[END_REF]. These pathways have been demonstrated to be overactivated in Fmr1-KO mice [START_REF] Bhattacharya | Genetic removal of p70 S6 kinase 1 corrects molecular, synaptic, and behavioral phenotypes in fragile X syndrome mice[END_REF][START_REF] Osterweil | Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome[END_REF][START_REF] Hou | Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is required for metabotropic glutamate receptor-dependent long-term depression[END_REF][START_REF] Gantois | Metformin ameliorates core deficits in a mouse model of fragile X syndrome[END_REF]. In agreement with a neuronal DAG increase in FXS condition, we showed that PKC is overactivated in Fmr1-KO neurons (Fig. 1A). Overall, the excess of DAG resulting from the lack of DGKk activity in Fmr1-KO mouse could underlie the molecular mechanism leading to the excessive mGluRI signaling and the increase of local protein synthesis [START_REF] Tabet | Fragile X syndrome: Are signaling lipids the missing culprits?[END_REF][START_REF] Moine | Of local translation control and lipid signaling in neurons[END_REF]. Based on the hypothesis that DGK activity alteration could contribute to the severity of FXS condition, we postulated that increasing DGK activity could be beneficial for the patients. We tested the impact of DGK agonist pioglitazone (PGZ) on Fmr1-KO mouse model and showed that PGZ corrects its molecular and behavioral alterations. In cortical neuron cultures, PGZ normalized the overactivation of PKC and eIF4E (respectively first and downstream effectors of DAG signal transduction pathway) observed in Fmr1-KO compared to WT neurons (Fig. 1B,C). These (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 19 data suggest that PGZ is able to inhibits DAG-PKC signaling in neurons like in endothelial cells [START_REF] Verrier | PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase[END_REF]. PGZ demonstrated also the ability to reduce the protein synthesis rate in neurons cultures (Fig. 1D). Thus, PGZ appears to be able to counteract some of the well-established molecular alterations of Fmr1-KO neurons. The administration of PGZ via intraperitoneal injection resulted in a better concentration of the molecule in the brain compared to an administration through alimentation (3.7±0.9 vs 19.4±9 nM, Fig. 2), however we cannot exclude that a proportion of the molecule was degraded during the food pellet preparation process that involved heating at 60°C for several minutes. Subcutaneous pellets that enable slow diffusion of the molecule while avoiding repeated injections appeared to be inappropriate for targeting the brain, giving the lowest concentration in brain.

PGZ administered daily for 7 days prior the beginning of behavioral tests and continued during the tests for a total of 19 days at 20 mg/kg/day (corresponding to approximately half the maximum recommended human oral dose of 45 mg based on mg/m 2 , Takeda Inc.) was able to correct the elevated level of main DAG species in the brain of Fmr1-KO mouse (Fig. 4) in agreement with its DGK agonist effect [START_REF] Verrier | PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase[END_REF]. PGZ normalized the observed behavioral alterations of the Fmr1-KO model. Notably, the recognition of novel object after 24h that was absent in Fmr1-KO was reestablished in Fmr1-KO-treated mice (Fig. 3A), suggesting that PGZ had effect on long term memory deficit of the FXS model. Reciprocal social interaction, stereotypical behavior (digging) and grooming were all normalized (Fig. 3B-D). We did not observe influence of genotype on locomotor activity but unexpectedly PGZ decreased the distance traveled and rearing numbers in both genotype, suggesting a possible adverse effect of treatment on voluntary locomotor activity (Fig. S2A). Whether linked or not with this effect, PGZ had an influence on free body fluid retention, this is a well-known edema causing effect of the molecule due to renal sodium (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 20 retention and vascular hyperpermeability [START_REF] Duan | Peroxisome proliferator-activated receptorgamma-mediated effects in the vasculature[END_REF]. However, the physical performances as assessed with rotarod test were not affected.

PGZ inhibits DAG-PKC signaling by activating DGK enzymes [START_REF] Verrier | PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase[END_REF], but the mechanism of this activation is still unclear. PGZ can act by PPAR gamma dependent and independent pathways [START_REF] Turturro | Troglitazone and pioglitazone interactions via PPAR-gamma-independent and -dependent pathways in regulating physiological responses in renal tubule-derived cell lines[END_REF]. In PGZ-treated neuron cultures and in brain extracts of PGZ-treated mice, no change was observed on the transcript level of DGK enzymes (Fig. S5) suggesting that in neurons, unlike in endothelial cells [START_REF] Verrier | PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase[END_REF], PGZ mediates DGK activity in a PPAR gamma independent manner. This is in agreement with the fact that PPAR gamma is highly expressed in adipose tissues [START_REF] Kliewer | Differential expression and activation of a family of murine peroxisome proliferator-activated receptors[END_REF] but very poorly expressed in brain [START_REF] Fagerberg | Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics[END_REF]. The exact mechanism of PGZ action in the brain remains thus to be understood.

An augmentation of cerebral DAG has been observed to coincide with the appearance of mild cognitive impairment in Alzheimer's (AD) (55), Parkinson's and Lewy Body disease [START_REF] Wood | Augmented frontal cortex diacylglycerol levels in Parkinson's disease and Lewy Body Disease[END_REF] and this increase has been proposed as a triggering factor of neuroinflammation [START_REF] Wood | Diacylglycerols as biomarkers of sustained immune activation in Proteinopathies associated with dementia[END_REF]. PGZ has established anti-inflammatory effects and had been shown to have positive cognitive effects on mouse models of AD [START_REF] Masciopinto | Effects of long-term treatment with pioglitazone on cognition and glucose metabolism of PS1-KI, 3xTg-AD, and wild-type mice[END_REF][START_REF] Searcy | Long-term pioglitazone treatment improves learning and attenuates pathological markers in a mouse model of Alzheimer's disease[END_REF], that like the FXS model have aberrantly elevated glutamatergic neurotransmission [START_REF] Lipton | Pathologically activated therapeutics for neuroprotection[END_REF]. Neuroinflammation has been evoked to play a role in FXS and other form of ID and autism [START_REF] Marco | Neuro-Inflammatory Mechanisms in Developmental Disorders Associated with Intellectual Disability and Autism Spectrum Disorder: A Neuro-Immune Perspective[END_REF]. In fact, GSK3, a strong promoter of inflammation in various tissues [START_REF] Jope | Glycogen synthase kinase-3 (GSK3): inflammation, diseases, and therapeutics[END_REF] has been shown to be overactivated in Fmr1-KO brain [START_REF] Min | Elevated glycogen synthase kinase-3 activity in Fragile X mice: key metabolic regulator with evidence for treatment potential[END_REF]. GSK3 activity has been demonstrated to be trigered by DAG/PA balance, notably by its impact on PI4P5-kinase to activate AKT/phosphatidynositol 3-kinase (PI3K) controlling GSK3 activity status [START_REF] Jenkins | Type I phosphatidylinositol 4-phosphate 5kinase isoforms are specifically stimulated by phosphatidic acid[END_REF][START_REF] Frame | A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation[END_REF]. Moreover, knock-out of DGKß, an isozyme of DGKk also expressed in brain, leads to an increase of GSK3ß activity proposed to contribute to the hyperactivity and reduced anxiety observed in the model [START_REF] Kakefuda | Diacylglycerol kinase beta knockout mice exhibit lithium-sensitive behavioral abnormalities[END_REF]. Thus, the loss of DGKk activity could be also responsible for this type of neuroinflammation related phenotypes and PGZ may also contribute to interfere with these alterations.

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21

In conclusion, in this study we provide lines of evidence that pioglitazone, one of the most widely used US Food & Drug Administration (FDA)-approved anti-diabetic corrects main molecular and phenotypic deficits of the FXS Fmr1-KO model. To our knowledge, this is the first study to report that acting on the DAG signaling in the brain can correct the behavioral alterations associated with FXS. Upregulating DGK activity may thus represent a potential therapeutic strategy for the treatment of FXS.

Correspondence and requests for materials should be addressed to HM. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

  The test was performed at a luminosity of 70 +/-5 lux in the plexiglass square arena used for open field test (44 x 44cm x 16 cm, Panlab Harvard apparatus IR ACTIMETER, Bioseb), considered as a habituation session 24h before training session. First part of the test consisted of a learning task. Two identical objects were presented to animals. Marbles and dices used for the test were randomly placed at defined objects positions. Exploratory activity was recorded for 10min. 24 hours later, second part of the test consisted of a memory recall. Two

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 11 objects were presented to animals at the same random position (left/right) with a new object replacing one of the learning session objects (next/familiar). Exploratory activity was recorded for 10min. For each session, sniffing time spent on objects and numbers of sniffing contacts were manually scored, defined as <2 cm distance between the nose and the object. Memory capacity was defined by calculated discrimination index (percentage of exploration of the novel object) as the proportion of time that animals spent investigating the novel object minus the proportion spent investigating the familiar one in the testing period: Discrimination Index = [(Novel Object Exploration Time -Familiar Object Exploration Time)/ Total Exploration Time] × 100.

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint 12 exploratory and care activities. Number of rearing or digging events and grooming were scored.Free sociabilitySociability was performed at 70 +/-5 lux in home-cage (GM 500 techniplast, floor area 501 cm²) for 10min. For the test two animals were isolated in a new home cage with sawdust bedding for 10min. Two social partners were conspecific of the same group (genotype, treatment), housed in same conditions (4-5 individuals per cages) but were non-siblings and housed in different cages. Number of contacts and times spent in contact were scored for affiliative behaviors (sniffing or touch any part of the body, allogrooming, followings or traversing the partner's body by crawling over/under it) and aggressive behaviors (attacks, offensive or defensive postures, tail rattling).

Figure 1 :

 1 Figure 1: Pioglitazone corrects the excessive downstream DAG signaling in Fmr1-KO

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted September 22, 2020. ; https://doi.org/10.1101/2020.09.22.301762 doi: bioRxiv preprint30GAPDH was used as a loading control. For quantification, the phospho-protein signal was normalized first against PKCa protein signal and is presented relative to the signal of WT neurons (n = 6 embryos in each group). B) PGZ(10 µM, 1h) normalizes PKC phosphorylation of Fmr1-KO neurons. Representative immunoblots of lysates from WT and Fmr1-KO cortical neurons (2 biological replicates for WT and 4 for Fmr1-KO are shown). Quantifications were made as in A with n ≥ 6 embryos in each group. C) PGZ (10 µM, 1h) normalizes eIF4E phosphorylation of Fmr1-KO neurons. Representative immunoblots of lysates from WT and Fmr1-KO cortical neurons (3 biological replicates for WT and Fmr1-KO are shown). Quantifications of p-eIF4E signal were normalized first against GAPDH protein signal and are presented relative to the signal of WT with n = ≥ 5 embryos in each group. D) PGZ reduces protein synthesis rate in WT and Fmr1-KO neurons. Representative immunoblots of puromycin-labelled neuron lysates from WT and Fmr1-KO cortical neurons treated with pioglitazone for 1h at indicated doses. Quantifications of puromycin signal were normalized first against GAPDH protein signal and are presented relative to the signal of WT with n = 2 embryos. E) PGZ (24h) normalizes S6 phosphorylation of Fmr1-KO neurons. Representative immunoblots of lysates from WT and Fmr1-KO cortical neurons. Quantifications of p-S6 signal were normalized as in C with n = ≥ 3 embryos in each group. In A-E, each point represents data from an individual neuron culture, all values shown as mean ± s.e.m. ***p< 0.001, **p< 0.01, *p< 0.05 vs Wt, ## p< 0.01, ### p< 0.001, #### p< 0.0001 vs Fmr1-KO untreated; ns, not significant; in A calculated by Student test and in B-E by one-way ANOVA with Tukey's multiple comparison test.

Figure 2 :

 2 Figure 2: Pioglitazone concentration in total brain extracts. PGZ concentration (nM) was

Figure 3 :

 3 Figure 3: Pioglitazone corrects main phenotypes of Fmr1-KO mouse. A) Novel object

Figure 4 :Figure S2 :

 4S2 Figure 4: Pioglitazone corrects elevated DAG level in brain of Fmr1-KO mouse. Measure

Figure S3 :

 S3 Figure S3: Influence of pioglitazone on novel object recognition, rotarod, prepulse

Figure S4 :

 S4 Figure S4: Influence of pioglitazone on weight and body composition. A) Weight

Figure S5 :

 S5 Figure S5: Influence of pioglitazone on DGK transcripts level in total brain extracts and

  acquisition was performed with the MS Workstation 8 software and data analysis with the MS Data Review software. Absolute quantifications were achieved by comparison of sample signals with doseresponse curves established with pure compounds. The MS/MS MRM transitions were (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2020.09.22.301762	this version posted September 22, 2020.	The copyright holder for this preprint
	357>134.10 and 357> 339 for the pioglitazone and 358>135 and 358>341.2 for the
	rosiglitazone.
					7

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2020.09.22.301762	this version posted September 22, 2020.	The copyright holder for this preprint
					A was
	ACN/MeOH/H2O (19/19/2, v/v/v) +0.2% formic acid +0.028% NH4OH, and eluent B was
	isopropanol +0.2% formic acid +0.028% NH4OH. The gradient elution program was 0-5min,
	15% (v/v) B; 5-35min, 15-40% B; 35-40 min, 80% B; and 40-55 min, 15% B. The flow rate
	was 40 μL/min, and 3-μL sample volumes were injected. The relative levels of DAG species
	were determined by measuring the area under the peak, determined by using MultiQuant
	software (Version 2.1; ABSciex) and normalizing to the area of the DAG internal standard.

(

Acknowledgements

We thank Dr. F. Sakane, Dr. R. Willemsen, Dr N. Vitale, Dr F. Riet for materials and discussions, N. Banquart-Ott and C. Nahy for mice care. PGZ analyses were performed at the Plant Imaging & Mass Spectrometry, PIMS, CNRS Strasbourg. We are very grateful to all (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. colleagues of the team for discussions and suggestions. The DAG analyses were performed at the Metabolome Facility-MetaboHUB (ANR-11-INBS-0010). This work was supported by ANR-18-CE12-0002-01 and Fondation Jérôme Lejeune funding to HM, by FRAXA Foundation post-doc fellow to AG and by Fond Paul Mandel to KH. This study was also supported by ANR-10-LABX-0030-INRT under the frame program Investissements d'Avenir ANR-10-IDEX-0002-02.

Author contributions

AG and KH performed most experiments and analyzed the data. DH LS and JZ performed PGZ mass spec analyses. LF performed diacylglycerol analyses. BZ, EF, assisted with molecular biology work. AD and YH helped with behavioral analyses. HM supervised the study and wrote the manuscript with AG, KH, BZ. All authors read the manuscript and approved the content.

Competing interests

The authors declare no competing financial interests.

Supplementary materials


Kewin Desjardins
email: kewin.desjardins@synchrotron-soleil.fr

Kadda Medjoubi

Pascal Mercère

Paulo Da Silva

Horia Popescu

Roland Gaudemer

Maurizio Sacchi

Adrien Besson

Rachid Belkhou

Stefan Stanescu

Sufal Swaraj

Stéphanie Pautard

Arafat Noureddine

Fabienne Orsini

Nicolas Jaouen

Backside-illuminated scientific CMOS detector for soft X-ray resonant scattering and ptychography

Keywords: X-ray detectors, sCMOS camera, 2D Soft

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In the last generation of synchrotron sources, the beamlines flux and beam coherence have been largely improved allowing to develop a large panel of sophisticated synchrotron radiation experiments. Nowadays, a lot of direct highly efficient detectors have been developed and are commercialized for hard X-ray application (Förster et al., 2019). They have demonstrated their high frame rates, high charge capacity, high efficiency and required high spatial resolution. However, a wide gap exists in the soft to tender X-ray domain (10 eV to 3 keV) where the experiments are not able to fully benefit from the performances offered by modern facilities. The detectors usually installed on the soft x-ray end-station are widely based on back-side illuminated CCD sensor such as the commercial cameras from Princeton© PI-MTE (https://www.princetoninstruments.com), GreatEye® GE-VAC (https://www.greateyes.de) or others equipped with cooled CCD-BSI. This kind of detector is compact enough, vacuum compatible (10 -9 mbar), presents performances largely demonstrated on laser based x-ray sources (Lambert et al, 2015), XFEL facilities (Pedersoli et al., 2011) or synchrotron beamlines (Chauleau et al, 2018). But these sensors suffers from severe readout speed limitation. Indeed, in order to limit the readout noise and so to guarantee a high signal to noise ratio allowing single photon sensitivity, the frame rate has to be limited to less than 1 frame per second. As an example, on SEXTANTS (Sacchi et al., 2013), the synchrotron SOLEIL's soft x-ray scattering beamline, they report a deadtime of 97.5%, due to the readout time in full-chip mode (typically 4 s over 0.1 s of X-ray collection), during Xray Fourier Transform Holography (FTH) experiments on the COMET end-station (Popescu et al, 2018). Nevertheless, some solutions have been implemented to tackle this problem: (i)

The fast-CCD (Denes et al., 2009) performs low readout noise with high frame rate (60 fps for 960 × 1920 pixels) by increasing the readout ports (ii) Other efforts have been done on Hybrid Pixel detectors (Jungmann-Smith et al., 2016, Wernecke et al. 2013) by decreasing the electronic noise level and optimizing the sensor entrance window allowing to profit of very high frame rate of new ASIC. (iii) Spectroscopic detector dedicated as pnCCD [START_REF] Strüder | High Speed Imaging and Spectroscopy with Low Energy X-Rays[END_REF]] could also be cited. (iv) the development of CMOS based sensor such Silicon-On-Insulator (SOI) technology (Arai et al, 2010) or DEPFET sensors [START_REF] Porro | [END_REF] for very high frame rate soft X-ray FEL applications, the CMOS PERCIVAL detector (Wunderer et al., 2014) again under development by a consortium of synchrotrons or also some developments teams effort as the Pipper MAPS technologies recently published (El Bitar 2019).

Recently, a new generation of large Back-Side Illuminated scientific CMOS sensor (sCMOS-BSI) has been developed [START_REF] Ma | Image Sensors and Imaging Systems[END_REF] and supplied by GPIXEL® (https://gpixel.com).

Especially designed in order to optimize the efficiency delivered in the visible light range (> 95% @ 550°nm), and keeping the good performances of the first generation of sCMOS (low readout noise, large full well capacity and high frame rate), this new cost efficient 4 Mpx sensor appears to be well suited for the soft x-ray applications, and has been already characterized for several use as soft x-ray resonant scattering [START_REF] Desjardins | AIP Conference Proceedings[END_REF] or Xray imaging spectroscopy [START_REF] Wang | [END_REF]. In this paper, we describe the integration of this sCMOS-BSI into an in-house vacuum compatible camera and its characterization. The performances have been measured on the METROLOGIE beamline (Idir et al, 2006) and are reported in the following sections. In particular, we have characterized the sCMOS-BSI camera dynamics (dark noise, readout noise, spatial uniformity, linearity and overall gain), the soft X-ray Quantum Efficiency in the energy range between 30 to 2000 eV, as well as the spatial, energy resolution and the radiation tolerance. We also report on different tests using this sCMOS-BSI camera: coherent diffraction pattern from a 5 µm pinhole at 186 eV at the METROLOGIE beamline, a soft X-ray Resonant Scattering experiment from nanostructure Co/Cu multilayer at 700 eV on SEXTANTS beamline and several soft X-ray ptychography reconstruction also at 706 eV on HERMES beamline (Belkhou et al, 2015).

Materiel

sCMOS GSENSE400BSI characteristics (Signal to Noise ratio in X-ray domain)

The integrated and characterized sensor is the GSENSE400BSI (figure 1a), a scientific CMOS Back Side Illuminated from GPIXEL® (https://gpixel.com). This sCMOS-BSI sensor Following these specifications, the Signal to Noise Ratio (SNR) of the GSENSE400BSI, defined by equation 1 (Janesick, J. R., 2007, (with equation 3.11 revised as discussed in stackexchange web forum)], has been calculated the different modes and compared to the other similar sensors in the X-ray domain (Figure 2). where S is the signal in (e-) equal to 𝑄𝐸 × 𝑁 × E ⁄ 3.65 with QE is the Quantum Efficiency depending of the beam energy E, N the number of photon, 3.65 eV is the electron-hole pair creation energy in silicon, F the Fano factor (~0.12 for silicon),  readout &  dark the temporal noises & DSNU and PRNU the spatial noises as given in table 1.

The figure 2 shows an example of the SNR for a X-ray beam of 500 eV and its comparison of each sensor. The spatial noise is excluded here (PRNU = 0 and DSNU = 0). In this figure

we also represent the photon detection limit (at 500 eV) corresponding to the full well capacity (in photon/mm²) taking into account the pixel area (see table 2 for all details). Now considering the HDR mode (dark noise of 3 e-/s/px, readout noise of 2 e-rms and FWC = 30 ke-) and the theoretical QE (as describe in the next paragraph, see §3.2), the limit of detection (SNR = 1) and the pixel saturation of the GSENSE400BSI sensor can be evaluated and the dynamic range (DR=FWC/limit) can be estimated in function of the beam energy (figure 3)

.

Figure 3 : Theoretical dynamic range of the GSENSE400BSI (HDR mode) considering the X-ray photon limits of detection (SNR=1) and the X-ray saturation limit (diamond red line).

X-ray camera design and software integration

In order to easily evaluate and integrate the sCMOS GENSE400BSI sensor on the soft Xray beamlines of SOLEIL synchrotron, we have adapted a TUCSEN® Dhyana95 camera, which has been totally disassembled and reassembled into a home-made mechanics ( Figure 4), to make it compatible as much as possible in vacuum environment (10 -6 mbar).

It can be noted that the components vacuum compatibility (cables, cooling, PCB board, …)

have not been the subject of in-depth studies for this first prototype assembly, whereas it is a crucial technical point which must be taken into account for the future detector that will be permanently installed on SOLEIL beamlines. Indeed, as seen during our series of tests, the cooled sensor could be quickly contaminated which could degrade its efficiency (Deiries et al, 2016). Then, following components are located in vacuum: sensor, sensor board, DAQ board (FPGA, power supply, data transfer, …) and a water-cooling circuit (> 12°C) has been made to maintain an optimal temperature of both the sensor (-20°C with 2 stages of Peltier cooler) and the electronic components. As typically done for such soft X-ray experiment, the To transfer and to record the images, this camera is connected via a USB3 interface through a dedicated UHV USB3 feedthrough to a classical Windows 64bit computer equipped with a Tango LIMA device (https://www.tango-controls.org) based on TUCAM API from TUCSEN®. Then, this camera keeps the same characteristics as the initial camera in term of SNR, readout speed (24Hz full frame), cooling and synchronization capacities.

Characterization tests results and discussion

X-ray experimental setup

As it has been already presented in the first DhyanaX prototype setup [START_REF] Desjardins | AIP Conference Proceedings[END_REF], this X-ray camera has been mounted in the reflectometer of the XUV branch of the METROLOGIE beamline at SOLEIL synchrotron. The beamline allows to select a photon beam in the soft X-ray range (30 eV -2000 eV) using several combination of gratings and mirrors to optimize the beamline transmission and energy resolution and a series of thin filters llowing to reduce the harmonics contamination in each configuration. The beam size could also be easily adjusted and a calibrated photodiodes (IRD® AXUV100, https://optodiode.com) can be used measuring, with less than 10% of uncertainty, the incident X-ray beam flux at each energy. With this configuration, the QE of two versions of GSENSE400BSI has been measured ( §3.2) over the full soft X-ray range. In addition, to characterize fully the detector, we have measured the spatial resolution at 1500 eV ( §3.3), the energy resolution ( §3.3) as well the sensor radiation hardness ( §3.4).

GSENSE400BSI soft X-ray Efficiency

Two versions of sCMOS GSENSE400BSI versions have been evaluated: the 𝑄𝐸 𝑚𝑜𝑑𝑒𝑙 (𝐸) = 𝑒 -𝑧 𝑆𝑖𝑂 ×𝜇 𝑆𝑖𝑂 (𝐸) × 𝑒 -𝑧 𝑆𝑖𝑁 ×𝜇 𝑆𝑖𝑂 (𝐸) × 𝑒 -𝑧 𝑛𝑒𝑢𝑡𝑟𝑎𝑙 ×𝜇 𝑆𝑖 (𝐸)

× (1 -𝑒 -𝑒 -𝑧 𝑑𝑒𝑝𝑙𝑒𝑡𝑒𝑑 ×𝜇 𝑆𝑖𝑂 (𝐸) )

(

where the z SiO and z SiN are the thickness material of the AR coating, 𝑧 𝑛𝑒𝑢𝑡𝑟𝑎𝑙 and z depleted are the thickness of the non-sensitive and sensitive silicon layer respectively. μSiO, μSiN and μSi are the attenuation coefficient for these different elements. To fit with the QE measurement results, the different parameters of the model (Equation 2)

have been adjusted. For the GSENSE400BSI-TVISB, the Silicon oxide thickness has been increased to 30 nm certainly due to the contamination and oxidation at its surface, the dead layer has been adjusted to 60 nm and the Si-Epi to 11 µm. Whereas the GSNESE400BSI-GP model fit correctly with the sensor parameters with a dead layer of 45 nm and a Si-Epi around 4 µm (and no-AR coating layer). Both measurements and models are reported in the figure 5.

Our result shows that the QE of the classical GSENSE400BSI is highly sufficient for a wide range of high flux synchrotron applications. In order to illustrate its low energy efficiency, a diffracted pattern image form a 5 µm diameter pinhole has been performed at an energy beam of 186 eV with the sCMOS GSENSE400BSI-TVSIB and the result is showed in figure 6. With twice the efficiency around 300 eV, the no-ARc sensor (GP version) could be a better choice for the so-called water window's region that include C, N and O K edge, but it should be noted that it will be at the price of using the thin Si-Epi layer sensor more sensible to radiation damage, as it will be shown in paragraph 3.4.

GSENSE400BSI spatial & energy resolution

The sCMOS GSENSEBSI400-TVISB spatial resolution has been checked using the ISO A 10% modulation transfer contrast is reached around 24 lp/mm, corresponding to a halfpitch resolution of 21 μm, in good agreement with the sensor pixel size (11 µm²). This resolution could be degraded for lower X-ray energy due to the lateral charge diffusion within the thick Epi-Silicon (~10 µm) and the neutral layer, but it was not the main object of our in this work. However, this effect will be discussed (figure 8b) in the next paragraph where we described the sensor energy resolution.

In order to characterize the energy resolution of the detector, a GSENSE400BSI-GP sensor has been exposed by a small part (out of focus) monochromatic X-ray beam of METROLOGIE beamline with very low flux (no more than 10 6 ph/s) in order to collect images of single isolated events. For each energy, a large series of cropped images (100 × 2048 pixels) has been acquired with the HDR mode and for a short exposure (20 µs, ~ 300 fps). This type of measurement, widely used in X-ray Spectroscopy Imaging and X-ray Spectroscopy astronomy (Lung et al., 1984) allows to work similarly as a counting detector with few photons per pixel per second. Then, for every dark-corrected image recorded, the single and isolated photon events (highlighted by blue circles in figure 8a) have been extracted and their amplitude cumulated to obtain a spectrum. This algorithm was carried out using in a MATLAB script isolated a pixel if its value is 95% greater than the sum of the 8 neighboring pixels. The result is displayed in figure 8b in which we superposed the normalized spectrums for energy from 100 eV to 1500 eV. In order to convert the spectra in eV the overall gain of 0.67 ADU/eV, calculated from this measurement, has been applied. The energy resolution is estimated by the full width at half maximum (FWHM) of the energy peaks [START_REF] Janesick | Scientific Charge-Coupled Devices[END_REF] and given in the figure 9 where the theoretical limitation given by the equation 3 below is also added for comparison:

𝐹𝑊𝐻𝑀 (𝑒𝑉) = 2.35 × 3.65 × √𝐹 × 𝐸/3.65 + 𝜎 𝑠𝐶𝑀𝑂𝑆 2 (3)
where F is the Fano factor (~0.1 for silicon), E the photon energy, 3.65 eV is the electronhole pair creation energy for X-ray absorption in silicon and 𝜎 𝑠𝐶𝑀𝑂𝑆 2 is the quadratic sum of noises of the sCMOS camera. We find an energy resolution that is slightly higher of 20 eV than the theoretical one, due to an excess noise in this camera prototype (with readout noise > 2 e-rms and higher offset and gain fixed-pattern noise). Furthermore, the sensor cooling was set to 16°C (only water cooling was operated and the Peltier stage was deactivated). In figure 9 is superposed a FWHM model fitting the datas with 𝜎 𝑠𝐶𝑀𝑂𝑆 2 of 6 e-rms. Also, the noise floor, represented in figure 8 by the tail on the energy peaks is reached for energy below 200 eV. As stated above, the effect is attributed to the spread charge created close to the surface into the thick Si and particularly in the non-depleted region.

GSENSE400BSI Radiation hardness

Considering the typical requirement for an X-ray detector needed at SOLEIL with energy of photon between 40 to 2000 eV and a monochromatic flux largely exceeded 10 12 ph/s/0.01%BW, radiation hardness has been evaluated on both sensors on the METROLOGIE beamline for different energies and X-ray fluences. The GSENSEBSI400-TVISB sensor has been locally exposed to a direct, collimated (< 200 × 200 µm²) and monochromatic X-ray beam of 160 eV, 560 eV, 1000 eV and 1600 eV and the GSENSE400BSI-GP only by a lower energy X-ray beam of 80 eV. For each energy, the delivered X dose has been increased by increasing the time of irradiation from few seconds to several hours (figure 10.a). The absorbed dose (D), i.e. the amount of energy absorbed per unit of mass of a given material (Ravotti, 2018), is basically calculated by using the following equation :

𝐷(𝑧) = μ 𝜌 × Φ/𝐴 × 𝑡 × 𝐸 (4)
with Φ is the considered X-ray photon flux, A is the surface exposed by the monochromatic X-ray beam with energy E, / the mass energy absorption coefficient (of Si) and t the exposure time.

The table 3 gives the absorbed dose evaluated on both the surface and the electronic layers of the sensor for different incident and different energies. We select characteristic energies over the wide soft x-ray energy range that allows to evaluate the radiation damage at the different layers on the sensor's thickness. Indeed, as shown in figure 11, the low x-ray photons are largely absorbed on the thin thickness of Epi-Si when the X-ray photons above 1000 eV goes through the Epi-Si and reach the electronic layer (figure 1b). Also, the photon beam at 80 eV could penetrate the thinner Epi-Si of the GSENSE400BSI-GP (4 µm) with a transmission about 0.1 %, against below 10 -5 % for the GSENSE400SI-TVISB (10 µm). As it can be found elsewhere (Clarke, 1994, Hancock et al., 1997), one of the radiation effect on the sensor manifests as an increased leakage current (pixel offset level), consequently we have compared the dark image after irradiations to the original one for the same exposure time, as showed in figure 10b. The mechanisms of these effects are complex and are not studied here but the results extracted from the figure 10.b are discussed. As described above, the low X-ray energies irradiation series (160 eV and 560 eV) with an absorbed dose up to 1

MGy into the surface layers of the GSENSE400TVSIB has no impact on the pixels offset level while the high energies X-ray irradiation series (1000 eV and 1600 eV) showed a significant increase of electronic offset level (for a lower dose) on the area irradiated (white spots on the right side of the dark image in figure 10.b). For these energies, the photons not absorbed by Epi-Si could penetrate deeply in the oxide and electronic layers of the CMOS sensor giving rise to a relatively important and persistent damage on the pixel (offset dark level remains high after few weeks at room temperature). It was similarly on the GSNES400BSI-GP with the 80 eV beam impact.

Then, the pixels dark level evolution (I dark after irration. -I dark0 ) has been compared to the dose on electronic layer for all irradiation series to extract an estimation of the limit of dose acceptable (figure12 by the sensor. The dark level increase, due to the dose, could be fitted with an empirical formula as:

𝐼 𝑑𝑎𝑟𝑘 -𝐼 𝑑𝑎𝑟𝑘0 = { 0, 𝐷 < 𝐷 𝑡ℎ 𝐾 × 𝑙𝑜𝑔 10 ( 𝐷 𝐷 𝑡ℎ
) , 𝐷 ≥ 𝐷 𝑡ℎ (5) where D is the dose on the electronic layer (equation 4), D th is the dose threshold and K a proportionality factor given the offset pixel evolution per (ADU/decade).

The fitted curve, in figure 12.a, for the two high energies measurement showed a limitation around D th = 200 Gy (20 kRad) and K = 125 ADU/dec. This dose threshold seems to be confirmed on the series of measurement performed on the thinner GSENSE400BSI-GP but with a higher dark evolution proportionality factor around 4000 ADU/dec, in figure 12.b.

If the dose limit seems not very high, it's very difficult to compare with other similar devices as this measurement is not easy to do or easy to exploit and so rarely published, nevertheless,

Experiments performed on Soft X-ray beamlines using the sCMOS GSENSE400BSI

@ 1000 eV, Limit = 2.3 10 11 ph/s/mm² (or 2.6 10 6 ph/s/pixel)

Soft X-ray Resonant Magnetic Scattering

The GSENSE400BSI camera has been firstly installed on the RESOXS (Jaouen et al, 2004) end-station of the SEXTANTS beamline at SOLEIL. In order to illustrate the capabilities of the detector, a soft X-ray (400 eV to 2000 eV) Resonant Magnetic Scattering (XRMS) (Chauleau et al, 2018) experiment on magnetic samples exposed to the soft X-ray beam was performed.

As mentioned above in this paper, high resolution, low noise and high charge collection capacity are strongly required for this type of acquisition and are achieved up to now with a direct CCD detection. The beamline routinely uses with users the Back-Illuminated CCD PIMTE from Princeton Instrumentation (4Mpixels e2V CCD 42-40, pixel size 13.5 µm, QE>90% @700 eV, FWC=100 ke-, electronic noise of 10 e-rms with 0.22 Hz). In order to experimentally compare the performances of the sCMOS camera with this CCD camera, soft X-ray scattering pattern from known a lithography prepared assembly of Xm*Xm square Co/Cu multilayer sample have been acquired. Unfortunately, the GSENSE400BSI camera has not been installed at the exactly same position compared to the CCD (figure 15). While the sCMOS was placed 1 m downstream from the sample, the CCD was placed closer, at distance of 25 cm, which, considering the sensor area, limits the angular aperture from 1.5° (sCMOS) to 6.3° (CCD). Despite these nooptimized test conditions, two similar acquisitions have been performed on the both sensor and following these characteristics: exposure time of 200 ms, accumulation of 100 images, HDR mode for GSENSE400BSI and ADC 1 MHz mode for the CCD. The resulting images represented in angle (𝜃) are given in figure 16 for the same scale. The beam was focalized into the CCD position and its divergence is few mrad, these explain why the diffracted spots are much wider and more separated for the sCMOS than for the CCD image. To compare these images, the spatial dimensions are transposing to the scattering vector q (𝑞 = 4𝜋 sin(𝜃) /𝜆, where 𝜃 is the angle between the direct beam and the detector axes and 𝜆 is the wavelength of the beam). The superposition of the one direction integrated diffraction peaks is presented in figure 17. It shows the similar distribution with a nice superposition of the peaks position but with a scattered intensity slightly different,

particularly close to the central peak (order 0). As the beam focalization, the aperture, the pixel size and the QE are not the same, the magnitude scattered intensity are not easy to compare directly. We plan to have a series of future experiments in the near future to obtain more experimental results about efficiency and SNR to compare to this well-known CCD BSI.

As first conclusion, we can conclude that similar image from sCMOS could be obtained for a total acquisition time of 20 s compared to the 8 min needed to record 100 images with the actual CCD, meaning a total time saved up to a factor 24 (limited by the exposure time). This gain will change drastically the time of acquisition of an image using Fourier Transform Holography that is typically of 1 h nowadays to few minutes in the near future. It will be really an important gain for imaging experiment when we have to scan an external parameter (magnetic field, electric field, temperature) or even more important for time-resolved experiments (Bukin et al, 2016). The capability to acquire image with such frame rate will also dramatically expand the time window available at SEXTANTS for soft resonant magnetic X-ray Photo Correlation Spectroscopy (XPCS) only obtain recently at NSLS2 using a fast CCD (Chen et al, 2019). Lastly, it will be also a real gain for soft X-ray resonant ptychography in reflection geometry that is nowadays limited only by the coherent flux available and mechanical instabilities (Popescu et al, 2019). It is important also be noted that all these approaches will fully benefit, in terms of coherent flux and smaller beam size (using standard optics), from the new storage ring under development.

Soft X-ray ptychography reconstruction

In HERMES, a soft X-ray beamline dedicated for X-ray microscopy and equipped for Scanning Transmission X-ray Microscopy (STXM) at SOLEIL, a new dedicated set up for soft X-ray ptychography has been recently implemented to improve the spatial resolution capability of the beamline by replacing the 1D detector (scintillator photomultiplier tube, PMT) by the sCMOS GSENSE400BSI camera. So, the classical 2D sample scanning experiment consisting to measure the transmitted intensity is now consisting to measure the transmitted diffraction pattern recorded with the sCMOS camera.

In order to illustrate the capability of this new set-up, ptychography experiment was performed on a siemens star at 700 eV. During the experiment, diffraction patterns were collected by illuminating the sample at ? × ? positions on a rectangular grid with a grid spacing of ? nm. Thanks to the fast readout of the camera and its sensitivity, the experiment was performed in less than 10 min.

The figure 18 To confirm these nice performances obtained on easy construct able structure, several measured have been performed, the result obtained from magnetotatic bacteria (magnetosomes) is reported here. The images have been acquired using X-ray at 700 eV, the scan step is 20 nm. A region-of-interest (1024 px by 1024 px) has been selected on the sCMOS which allows to increase the frame rate up to 44 Hz (HRD mode) and so reduced the total acquisition to 10 mn. Again we presented, in the Figure 19

Summary and perspectives

A new camera based on the first world large production Back Side Illuminated scientific CMOS sensor GSENSE400BSI was developed and evaluated for soft X-ray application on several beamlines at SOLEIL synchrotron. The quantum efficiency is found to be sufficient for large field of application in the soft X-ray domain (QE > 50% for E > 400 eV) and the new no-ARC (antireflective layer) has already demonstrated a high quantum efficiency of > 90 % in the lower energy range and more importantly over the water window region. The readout noise (< 2 e-rms) and the full well capacity provide a good X-ray dynamical range and an interesting single photon detection capability without decrease of the readout speed performance. This sensor has a frame rate up to 48 Hz (on the standard mode) that allows to dramatically reducing the acquisition time for imaging application compared to the classical camera commonly used. The new version of acquisition board allows to upgrade the performance with a lower spatial noise with DSNU < 0.2e-and a PRNU of 0.3%. The soft Xray spatial and energy resolution has been confirmed with a limitation for low energy due to the low dead thickness and the thin Si-Epi layers.

As for similar synchrotron direct X-ray detector, the radiation hardness is a big challenge to keep good performances in the time with the high density of flux considered. The limit of dose of 200 Gy (20 kRad) and the corresponding fluence has been measured that will allow scientists to adapt the X-ray experimental conditions set-up in order to avoid damage of the sensor. A compact, cooled vacuum compatible camera prototype has been developed to be easily installed in the experimental end-stations of SEXTANTS and HERMES beamlines at SOLEIL. A first soft X-ray Resonant Scattering (REXS) acquisition from nanostructured multilayer sample has been performed on SEXTANTS beamline showing a good dynamical range with a total time saver of a factor 24 compare to their usual detector readout. A fully functional soft X-ray ptychography set-up based on this sCMOS camera has been used on the STXM end station at the HERMES beamline. Its performances and frame rate have been used to perform several reconstructions with spatial resolution down to ~6 nm.

Lastly, a new GSENSE400BSI sensor version combining a thicker Si-Epi with a high efficient process has been very recently tested by Harada team (Harada et al, 2020). It assures a very good efficiency (> 80 %) over a very large soft X-ray range and it will soon be massproduced by GPIXEL®. This new sensor will be very soon implemented on the second sCMOS HERMES camera to increase the set-up performances and particularly for the experiment at the so-called water window's carbon K-edge. Also, one other camera would be installed soon in the SEXTANTS COMET end-station in order to optimize the performance of acquisition time for magnetic imaging using Fourier Transform Holography.

Based on this first results and the scientist's community interest for this cost-efficient solution, a Canadian company specialist on high speed camera solution, Axis photonique [https://www.axis-photon.com] has started to produce this new soft X-ray sCMOS detector based on the SOLEIL prototype. Furthermore, others potential domains could be explored:

another synchrotron beamline application, as tender X-ray SAXS (> 1.2 keV) in SIRIUS beamline at SOLEIL, or for lower soft X-ray photon source based on femtosecond laser or for soft X-ray free electron laser (XFEL). In particular a test of our camera is already planned to take place in the next few months at FERMI.

To conclude, the first Back Illuminated sCMOS implementation reported in this paper could be considered as the beginning of a long series of new detector based on this technology.

is a 4

 4 PHOTOMETRICS®Prime95 (www.photometrics.com), the ANDOR® Sona (https://andor.oxinst.com) or others. These cameras take advantage of the sensor characteristic in term of readout noise (< 2 e-rms), relatively large Full Well Capacity (FWC up to 80 ke-), good performances in term of spatial noise (DSNU < 5 e-and PRNU < 1 %)

Figure 1 :

 1 Figure 1 : sCMOS GSENSE400BSI sensor (a) Sensor picture (Gpixel, http://gpixel.com) and (b) schematic cross section of the sensor

Figure 2 :

 2 Figure 2 : Signal to Noise Ratio estimation for GSENSE400BSI (Low Gain and High Gain), PIMTE CCDBSI (with ADC speed of 1MHz), PERCIVAL (Low Gain and High Gain) at 500 eV. The SNR of perfect detector is calculated for QE=1, noise = 0 eavec pixel size of 10 µm. The SNR of each detector are calculated from the details reported in table2and considering a dark noise given for 1 s of integration time. The saturation level of each compared sensors is given by dot, pentagon and star respectively for PERCIVAL, PIMTECCDBSI and GSENSE400BSI with a surface normalization.

  assembly has been designed to have a perfect tightness in parasite light with the possibility to add different visible filter (for example, carbon or alumina filter from Luxel®, https://luxel.com) in front of the sensor. It remains compact (10 cm × 11 cm × 12 cm) to fit with a vacuum chamber on beamline and their restrictive environment. Furthermore, an X-ray beam stop could be added in front of the camera and positioned with two linear piezo motorizations as shown in Figure 4 (Popescu et al 2019).

Figure 4 :

 4 Figure 4 : Mechanical design of GSENSE400BSI camera vacuum adaptation made at SOLEIL, so-called DhyanaX

  Figure 1 (b), is composed by a silicon epitaxial layer (Si-Epi) of 10 µm thick, 7 nm of SiO2followed by 63 nm Si3N4. This silicon nitride is on the top of the sensor, both layers serve as an anti-reflection coating (ARc) purpose and passivation layer (originally processed for visible applications). The second sensor tested has no anti-reflective coating (no-ARc) on the photo-sensitive area but has a thinner Si-Epi layer thickness around 4 µm. The method and measurement of the QE of the sensor have already been reported in the previous work(Desjardins 2019). The final result for both sensors is presented in figure5where the measurements have been superimposed with model results proposed in equation 2;

Figure 5 :

 5 Figure 5 : GSENSE400BSI-TVISB and GSENSE400BSI-GP Quantum Efficiency measurements compared to a theoretical model given by AR coating and Si-Epi absorption.

Figure 6 :

 6 Figure 6 : Circular integration of pinhole diffraction image from sCMOS GSENSEBSI400 obtained at 186 eV on METROLOGIE Beamline (100 accumulations of 100 ms dark-corrected images in HDR mode).

  knife slanted edge method (International Organization for Standardization [ISO], 2000). A silicon nitride support films has been used as edge slightly tilted (the edge angle was about 10° off of the pixel array alignment) and illuminated with X-ray beam at 1500 eV. The Modulation Transfer Function (MTF) has been calculated from cropped dark-corrected image (figure7) carried out by using sfrmat3 Matlab script[START_REF] Burns | SFRmat3 Script[END_REF]. The result is given in pair of line per mm in figure7.

Figure 7 :

 7 Figure 7 : MTF calculation from sCMOS GSENSEBSI400 slanted edge image at 1500 eV on METROLOGIE Beamline (1000 accumulation of dark corrected images of 100 ms).

Figure 8 :

 8 Figure 8 : (a) Example of image analyzed for X-ray energy beam of 1000 eV (blue circles are the pixel extracted). (b) Spectrums of single-event frequencies for X-ray photon energy of 100-1500 eV. The spectrums for 400 eV to 1500 eV have been voluntary cropped in low-energy range (down to 200 eV).

Figure 9 :

 9 Figure 9 : X-ray energy resolution of GSENSE400BSI-GP measurement results compared to the Fano noise limit and to the fit FHWM with σ sCMOS = 6 e-.

Figure 10 :

 10 Figure 10 : (a) Schematic representation of X-ray irradiation of GSENSEBSI400-TSVIB for 160 eV, 560 eV 1000 eV and 1600 eV (b) Part of GSENSEBSI400-TSVIB dark image (300 px by 1000 px) after irradiation (contrast voluntary increased)

Figure 11 :

 11 Figure 11 : Theoretical transmission versus the Si thickness for X-ray photon of 160 eV, 560 eV, 1000 eV and 1600 eV through the GSENSEBSI400-TVISB and of 80 eV through the GSENSEBSI400-GP.

Figure 12 :

 12 Figure 12 : Radiation tolerance evaluation of (a) sCMOS GSENSE400BSI-TVSIB and (b) sCMOS GENSE400BSI-GP by excess dark measurement after soft X-ray irradiation with beam of 560 eV, 1000 eV and 1600 eV and 80 eV, respectively. In dashed and red curves are the empirical model (equation 5).

  e2v reports a radiation tolerance of 10 kRad (100 Gy) on the classical CCD design [datasheet du CCD40-42, D. Burt et al, 2009]. From this value (D th = 200 Gy), we represent in figure 13 an estimation of the limit of fluence (number of photons/unit area/time) to avoid non reversible damage of the two sensors tested here.

Figure 13 :

 13 Figure 13 : Limit of fluence [ph/s/mm²] calculated for a limit the dose of 200 Gy on electronic layer of a GSENSE400BSI-TVSIB before damaging.

Figure 14 :

 14 Figure 14 : Exposure time limit calculated for a limit the dose of 200 Gy on electronic layer of a GSENSE400BSI-TVSIB (blue) and GSENSE400BSI-GP (red) before damaging.

Figure 15 :

 15 Figure 15 : Exposure time limit calculated for a limit the dose of 200 Gy with a flux of 10 s ph/s/(11 x 11 µm²) on electronic layer of a GSENSE400BSI.

Figure 16 :

 16 Figure 16 : SEXTANTS beamline synoptic for GSENSE400BSI camera test versus CCD installed. SEM image of the Co/Cu nanostrured sample of 530x1220nm rectange.

Figure 17 :

 17 Figure 17 : Dark corrected images of soft X-ray scattering from Co/Cu multilayer sample at 767 eV acquired with (a) the sCMOS GSENSE400BSI camera placed at 1 m behind the sample and (b) the PIMTE camera placed close to the sample (25 cm). Both images correspond to the accumulation of 100 images of 200 ms.

Figure 18 :

 18 Figure 18 : Superposition and normalized the sum of the pixels in central region of the images (figure 16) obtained with CCD camera (blue line) and sCMOS camera (red line) for X-ray energy photon of 767eV versus the scattering vector.

Figure 19 :

 19 Figure 19 : (a) STXM image with beam size spot of XXXXX and (b) Ptychography reconstruction of the Siemens star. (c) Example of a speckle pattern.

  .a, the lower resolution STXM classical image obtained with PMT while in the Figure.19.b, we showed the reconstructed ptychography image performed with the ePIE method implemented in a MATLAB script developed by the NANOSCOPIUM beamline[Medjoubi et al, 2018] and used by several SOLEIL beamlines. This reconstruction allows to clearly observe the bacteria (diameter of ? nm) with a very small space between two adjacent magnetosomes.

Figure 20 :

 20 Figure 20 : (a) Classical STXM image of magnetosomes at 706 eV. (b) Ptychography reconstruction of magnetosome. (c) Example of a speckle pattern

Table 1 Electro

 1

		Symbol Value	ref
	Gain	K	Low gain, High gain or HDR mode
		fps	24 Hz Full frame (HDR)
	Frame Rate		48 Hz Full frame (STD)
	Readout architecture		Rolling Shutter	GPIXEL datasheet (www.gpixel.com)
	Pixel Size	px	11 x 11 µm²
	Sensor size		4M -2048 × 2048 pixels (22.5 mm × 22.5 mm)
	Exp. times	t	20 µs -10s
	Binning		No
	Readout noise Dark noise Full Well Capacity Spatial pixel offset noise Spatial pixel gain noise	 read  dark FWC DSNU PRNU	< 2 e-rms (HDR & HG) & < 45 e-rms (LG) > 3 e-/s/pix (-20°C) 30 ke-(HDR), 1700 e-(HG) & > 80 ke-(LG) < 5 e-< 1 %	TUCSEN Dhyana95 datasheet (www.tucsens.com) Wang et al., 2017 Desjardins et al, 2019

-optical characteristics of the GSENSE400BSI sensor specifications and TUCSEN Dhyana95 camera measurements results

table 2 and considering a dark noise given for 1 s of integration time. The saturation level of each compared sensors is given by dot, pentagon and star respectively for PERCIVAL, PIMTECCDBSI and GSENSE400BSI with a surface normalization.

	QE (500 eV)	0.85	~ 0.8 (unpublished)	0.5 up to 0.8 (see §3.2)
	Readout noise	10 e-rms (1MHz ADC)	1600 e-rms (lowest gain) 15 e-rms (highest gain)	2 e-rms (HG) 38 e-rms (LG)
	Dark noise	0.02 e-/s/pixel (-50°C)	Supposed low (-40°C) (unpublished)	3 e-/s/pixel (-20°C)
	FWC	temporal noise 100 ke-	shot noise ~ 4 Me-/pixel (lowest gain) spatial noise 80 ke-(LG) 30 ke-(HDR) ~ 2000 e-(highest gain) 1600 e-(HG)
		PI-MTE CCD BSI
	Detector	(datasheet PIMTE2048B) [http://www.princeton.c	PERCIVAL (BSI) (Khromova et al, 2016)	GSENSE400BSI (Desjardins et al, 2019)
		om]
	Pixel size	13.5 x 13.5 µm²	27 x 27 µm²	11 x 11 µm²

Table 3 . Soft X-ray irradiation details

 3

	Energy	Incident flux ( Φ 0 ) on the sensor	TID on surface 1	TID on electronic layer 2
		(Total exposure time)
	1 The TID (absorbed dose) on the surface is estimated from equation 4 considering the anti-reflection layers

thickness attenuation and with μ/ρ extracted from NIST FFAST database (https://physics.nist.gov) 2 The TID (absorbed dose) on the electronic layer is estimated from equation 4 considering the antireflective layer and Epi-Si thicknesses attenuation and with μ/ρ extracted from NIST FFAST database (https://physics.nist.gov)

Acknowledgments

The authors wish to acknowledge Hao Yuan and Adam Hitchcock of the Department of

Chemistry and Biology McMaster University (Ontario, Canada) for providing us the greath helpful with Pytochography reconstruction

We also would like to acknowledge Ye(Evan) Tian from TUCSEN and Xinyang Wang from GPIXEL for the great help sharing all needed information for this work. Finally, we are very grateful to Zhang Jiang of Argonne National Laboratory for sharing the Matlab toolbox on Xray refraction of matter [Jiang 2020].