Ursule Dumont
email: ursule.dumont@rmsb.u-bordeaux.fr

Stéphane Sanchez
email: stephane.sanchez@rmsb.u-bordeaux.fr

Benjamin Olivier
email: benjamin.olivier@rmsb.u-bordeaux.fr

Jean-François Chateil
email: jean-francois.chateil@chu-bordeaux.fr

Denis Deffieux
email: denis.deffieux@u-bordeaux.fr

Stéphane Quideau
email: stephane.quideau@u-bordeaux.fr

Luc Pellerin
email: luc.pellerin@unil.ch

Marie-Christine Beauvieux
email: marie-christine.beauvieux@chu-bordeaux.fr

Anne-Karine Bouzier- Sore
email: anne-karine.bouzier-sore@rmsb.u-bordeaux.fr

Hélène Roumes
email: helene.roumes@rmsb.u-bordeaux.fr

Maternal alcoholism and neonatal hypoxia-ischemia: neuroprotection by stilbenoid polyphenols

Keywords: neonatal hypoxia-ischemia, neuroprotection, polyphenols, magnetic resonance imaging, maternal supplementation, maternal alcoholism

The impact of maternal nutrition on neurodevelopment and neonatal neuroprotection is a research topic with increasing interest. Maternal diet can also have deleterious effects on fetal brain development. Fetal exposure to alcohol is responsible for poor neonatal global development, and may increase brain vulnerability to hypoxic-ischemic (HI) encephalopathy, one of the major causes of acute mortality and chronic neurological disability in newborns. Despite frequent prevention campaigns, about a quarter of women in the general population drinks alcohol during pregnancy and breastfeeding. This study was inspired by this alarming fact. Its aim was to evaluate the beneficial effects of maternal supplementation with two polyphenols during pregnancy and breastfeeding, on hypoxic-ischemic (HI) neonate rat brain damages, sensorimotor and cognitive impairments, in a context of moderate maternal alcoholism. Both stilbenoid polyphenols, transresveratrol (RSV -0.15 mg/kg/day), and its hydroxylated analog, trans-piceatannol (PIC -0.15 mg/kg/day), were administered in the drinking water, containing or not alcohol (0.5 g/kg/day). In a 7-day post-natal rat model of HI, our data showed that moderate maternal alcoholism does not increase brain lesion sizes measured by MRI but leads to higher motor impairments. RSV supplementation could not reverse the deleterious effects of HI coupled with maternal alcoholism.

However, PIC supplementation led to a recovery of all sensorimotor and cognitive functions. This neuroprotection was obtained with a dose of PIC corresponding to the consumption of a single passion fruit per day for a pregnant woman.

Highlights:

• Maternal alcoholism increases deficits induced by neonatal hypoxia-ischemia (HI).

• These impairments are counteracted by maternal supplementation with piceatannol.

• Doses used are equivalent to the piceatannol content of a single passion fruit.

• Piceatannol is neuroprotective in neonatal HI in a context of maternal alcoholism.

• Originality of the study: nutritional and transgenerational therapeutic approach.

Introduction

Neonatal hypoxia-ischemia (HI) is an important socio-sanitary problem as a major cause of morbidity and mortality in human newborns (1-6/1,000 births) with a high risk of chronic motor, behavioral and neurological deficits [START_REF] Robertson | School performance of survivors of neonatal encephalopathy associated with birth asphyxia at term[END_REF][START_REF] Shankaran | Acute neonatal morbidity and long-term central nervous system sequelae of perinatal asphyxia in term infants[END_REF] . HI is characterized by a cerebral deprivation of oxygen and glucose leading to cell death, the most affected being neurons [START_REF] Northington | Early neurodegeneration after hypoxia-ischemia in neonatal rat is necrosis while delayed neuronal death is apoptosis[END_REF][START_REF] Hilario | Cerebral blood flow and morphological changes after hypoxic-ischaemic injury in preterm lambs[END_REF] , which leads to sensorimotor deficits and mental retardation associated to learning abnormalities [START_REF] Robertson | Long-term follow-up of term neonates with perinatal asphyxia[END_REF][START_REF] Aridas | Detecting brain injury in neonatal hypoxic ischemic encephalopathy: Closing the gap between experimental and clinical research[END_REF] . Despite this major public health problem, the only current clinical treatment is hypothermia, which is thought to slow down neuronal cell death processes by reducing brain metabolism [START_REF] Hashimoto | Selective brain hypothermia protects against hypoxicischemic injury in newborn rats by reducing hydroxyl radical production[END_REF][START_REF] Gluckman | Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: Multicentre randomised trial[END_REF][START_REF] Shankaran | Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy[END_REF] . This unique therapeutic solution does not appear sufficiently successful (44-53% of infants die or suffer moderate to severe neurological disability after hypothermia treatment [START_REF] Wu | Erythropoietin: A novel therapy for hypoxic-ischaemic encephalopathy?[END_REF] ) and new therapeutic approaches must be developed to possibly reverse neonatal HI sequelae. In parallel to this neonatal public health problem, despite better communication about the risks, alcohol consumption in Western countries of pregnant women has not decreased [START_REF] Corrao | A meta-analysis of alcohol consumption and the risk of 15 diseases[END_REF] . In the USA, 1 out of 12 pregnant women drink alcohol [START_REF] Li | Fetal stress and programming of hypoxic/ischemic-sensitive phenotype in the neonatal brain: Mechanisms and possible interventions[END_REF] .

Maternal alcohol consumption during pregnancy and breastfeeding is well known to lead to deleterious effects on neurodevelopment. It represents the major cause of intellectual disability in neonates [START_REF] Abel | Fetal alcohol syndrome is now leading cause of mental retardation[END_REF] , a phenomenon called fetal alcohol syndrome, which incidence is 1/500 births [START_REF] Dan | fetal alcohol syndrome][END_REF] . In HI context, it has been shown in vitro, on primary neuronal cultures, that combination of ethanol, even at moderate doses, and hypoxia (0.01% ethanol during 24 h plus 0.1% O2 during 30 min) promote synergistically neuronal death [START_REF] Genetta | Low doses of ethanol and hypoxia administered together act synergistically to promote the death of cortical neurons[END_REF] . Therefore, neonatal HI in a maternal alcohol exposure context needs better investigations. We used the classical Rice-Vannuci rat HI model [START_REF] Rice | The influence of immaturity on hypoxic-ischemic brain damage in the rat[END_REF] , which briefly consists of a permanent unilateral occlusion of the common carotid artery, followed by a hypoxic episode, in 7-days old pups, an age that corresponds to a full-term newborn. This HI model induces large ipsilateral striatal, hippocampal and cortical injuries [START_REF] Ashwal | Comparison of two neonatal ischemic injury models using magnetic resonance imaging[END_REF] , leading to sensorimotor and cognitive deficits [START_REF] Lebedev | Neurological deficit and disturbances in higher nervous activity during modeling of perinatal hypoxic-ischemic damage to the central nervous system in rat pups[END_REF] .

The impact of maternal nutrition on neonatal neurodevelopment and on neonatal neuroprotection is a research topic with increasing interest [START_REF] Black | Broccoli sprout supplementation during pregnancy prevents brain injury in the newborn rat following placental insufficiency[END_REF][START_REF] Loren | Maternal dietary supplementation with pomegranate juice is neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury[END_REF] . Maternal dietary supplementation with ingestion of pomegranate juice (32, 16 or 8 µmol of polyphenols/day), which contains important quantities of polyphenols, was shown to lead to significant neuroprotection [START_REF] Loren | Maternal dietary supplementation with pomegranate juice is neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury[END_REF] . In another study, in a dam supplemented with a pomegranate polyphenol extract (4.8 mg polyphenols per day, orally administered during pregnancy and after delivery), a neuroprotective effect on neonatal brain injury after HI was also observed [START_REF] West | Pomegranate polyphenols and resveratrol protect the neonatal brain against hypoxic-ischemic injury[END_REF] . These authors also tested direct intraperitoneal trans-resveratrol (RSV) administration on pups. RSV injection (200 µg/kg or greater) reduced caspase-3 and calpain activation and could protect neonatal brain against HI injuries. RSV, notably found in grape skins and several types of nuts or roots is one of the most extensively studied polyphenols. It can cross the placenta [START_REF] Bourque | Maternal resveratrol treatment during pregnancy improves adverse fetal outcomes in a rat model of severe hypoxia[END_REF] and its maternal consumption during pregnancy is safe [START_REF] Williams | Safety studies conducted on high-purity trans-resveratrol in experimental animals[END_REF] . Although the therapeutic properties of RSV are well documented, its bioavailability is low (<1%). Its hydroxylated analogue, trans-piceatannol (PIC), found in large quantities in the seeds of passion fruit, has a two-fold higher bioavailability [START_REF] Kukreja | Therapeutic role of resveratrol and piceatannol in disease prevention[END_REF] . However, very few studies have been carried out with this compound and only one study has been conducted on neonatal HI [START_REF] Dumont | Maternal consumption of piceatannol: A nutritional neuroprotective strategy against hypoxia-ischemia in rat neonates[END_REF] . In this study, we show that PIC maternal supplementation (0.15 mg/kg/d) is neuroprotective by decreasing the incidence of HI lesion, decreasing the lesion volume and counteracting the deleterious effects on sensorimotor and cognitive abilities of pups.

The aim of the present work was to study the effect of a maternal supplementation with RSV or PIC on HI-pup brain damage in a context of maternal alcoholism. Pregnant and nursing dams were supplemented with RSV or PIC and/or ethanol and the Rice-Vannucci rat model was used to induce HI brain injury in their post-natal seven-day-old pups (P7). We evaluated in vivo neonate brain damages and their evolution with noninvasive magnetic resonance imaging (MRI). Cell death and brain edema were quantified in young rats for each maternal nutritional condition. Finally, motor and behavioral studies were carried out to evaluate the effect of polyphenols on cognitive function altered by HI event and maternal alcoholism.

Results

Piceatannol but not resveratrol reduces neonatal brain injuries after an HI episode, in a moderate maternal alcoholism context

Typical apparent diffusion coefficient (ADC) maps of Diffusion Weighted Image (DWI) of P7pup brains (3 h post-ligation) are presented in Fig. 1A. As expected, the sham group did not show any lesions. In all other groups, HI induces brain damages (Fig. 1A, hyposignal). HI injury affected more particularly the cortex, the hippocampus and the striatum. At P7, no difference in lesion volumes was detected between the different HI groups (Fig. 1B). At P9, pups that underwent an HI episode and whose dam was supplemented with both PIC and a moderate dose of ethanol (PIC/Eth group) had significantly lower lesion volumes than control HI pups (HIC group; pups with HI and coming from dam without any supplementation) and HI pups from dam supplemented with ethanol (Eth group) (8% ± 1% for PIC group vs 19% ± 4% and 18% ± 2% for HIC and Eth groups, respectively). DWI allows the basic measure of ADC. This value corresponds to the displacement rate of extracellular water molecules; the smaller, the more severe the cytotoxic edema. Typically, ADC values decrease within minutes of brain injury as a reflection of this cytotoxic edema. The change in ADC value after HI is a dynamic process and tends to return to higher values 24 to 48 h after the insult, a phenomenon called "pseudonormalization" [START_REF] Lodygensky | Application of magnetic resonance imaging in animal models of perinatal hypoxic-ischemic cerebral injury[END_REF] . ADC values at P7 were quantified in cortex, hippocampus and striatum (Fig. 1C). ADC significantly decreased in all HI groups. The severity of edema in the cortex and the hippocampus was significantly lower in the Eth and PIC/Eth groups compared to the other HI groups.

Neuronal death was evaluated in the penumbra region (cortex and striatum) by Nissl staining on P9-pups (Fig. 1D). For the cortical region, the HIC group presented the highest level of neuronal death compared to other HI groups (Fig. 1E). No significant difference was measured between Eth and RSV/Eth groups. However, less neuronal death was measured in the PIC/Eth group compared to the RSV/Eth group (7% ± 6% vs 21% ± 4%, respectively). In the striatal region, the PIC/Eth group presented the lowest level of neuronal death compared to other HI groups and both Eth and RSV/Eth groups presented the highest level of neuronal death.

Maternal polyphenol supplementation allows recovery of neonatal HI brain lesions detected by MRI despite moderate maternal alcoholism

At P30, 23 days after the HI insult, brain lesions were still observed by MRI in all pups in the HIC and Eth groups, whereas no more lesion was detected for 20% and 50% of pups in the RSV/Eth and the PIC/Eth groups, respectively (Fig. 2A). Pups in the HIC, Eth and RSV/Eth groups had larger lesion volumes compared to pups in the PIC/Eth group (Fig. 2B). Even if the incidence of brain damage was decreased by maternal RSV supplementation, RSV administration did not reduce lesion volumes (Fig. 2B, 31% ± 5%; 27% ± 3%; 24% ± 7%; 12% ± 6%, in HIC, Eth, RSV/Eth and PIC/Eth groups, respectively).

Piceatannol counteracts the deleterious sensorimotor and cognitive effects induced by neonatal HI, despite moderate maternal consumption of ethanol

Cortex and striatum, two structures involved in automatic motricity, were almost systematically damaged in our HI model.

The righting test was performed at P8, P10 and P12 to evaluate the impact of the different maternal supplementations on sensorimotor abilities and neurological reflexes after HI (Fig. 3A). Pup performances improved with time, reflecting a process of learning and habituation, in all groups except in the Eth group. Best performances were measured for sham pups and pups from the PIC/Eth group (no significant difference between these two groups). A delay in learning was observed for pups in the HIC and RSV/Eth groups compared to the ones in sham and PIC/Eth groups. Pups in the HIC and RSV/Eth groups showed intermediate performances between sham or PIC/Eth groups and Eth group, in which pups had the poorest performances.

The same pattern was observed in the negative geotaxis test. Pups from the Eth group showed the worst performances compared to the other HI groups and pups from the PIC/Eth group had identical abilities than the sham group (no significant difference between sham and PIC/Eth, whatever the considered test day) (Fig. 3B).

Since the HI model (unilateral carotid ligation) induces ipsilateral lesions, sensorimotor asymmetries were assessed using the adhesive removal test (Fig. 3C). This test allowed to discriminate between pups from the Eth group and all other pups since they had the poorest performances. The time to remove the adhesive was decreased with days, indicating that animals were learning the task, for all pups except the ones in the Eth group. Pups in the RSV/Eth group presented intermediate abilities while HIC and PIC/Eth groups performed as well as the sham group (no significant difference between sham, HIC and PIC/Eth groups).

Sensorimotor deficits were also assessed using the modified neurological severity score (mNSS) (Fig. 3D). Animals were evaluated at P24 (17 days post-HI). The mNSS score in sham and PIC/Eth groups was significantly lower than the one measured in HIC, Eth and RSV/Eth groups (0.38 ± 0.18; 0.83 ± 0.31; 4.86 ± 0.88; 5.0 ± 0.47 and 4.83 ± 1.24 for sham, PIC/Eth, HIC, Eth and RSV/Eth groups, respectively).

In our HI model, hippocampi were frequently injured. For this reason, the novel object recognition test was used to evaluate hippocampal-dependent long-term memory of pups at a later stage (Fig. 3E). Animals were tested at P45. Juvenile rats in the HIC and Eth groups presented a long-term memory deficit, whereas no difference was found between animals in the sham and PIC/Eth groups.

Rats in the RSV/Eth group presented an intermediate ability to discriminate the novel object (discrimination index: 0.77 ± 0.04; 0.47 ± 0.09; 0.41 ± 0.04; 0.58 ± 0.09; 0.66 ± 0.02 for sham, HIC, Eth, RSV/Eth and PIC/Eth, respectively).

Discussion

Neonatal HI caused by oxygen and glucose deprivation induces an energetic failure and a decrease in ATP, which in turn leads to a reduced sodium-potassium ATP pump activity and membrane depolarisation. The loss of membrane polarity leads to glutamate efflux and calcium influx [START_REF] Salter | Nmda receptors are expressed in developing oligodendrocyte processes and mediate injury[END_REF][START_REF] Karadottir | Neurotransmitter receptors in the life and death of oligodendrocytes[END_REF] .

Calcium is responsible for the activation of several enzymes such as proteases, endonucleases and nitric oxide synthase [START_REF] Mitani | Stimulation of glut-1 glucose transporter expression in response to exposure to calcium ionophore a-23187[END_REF] . Therefore, the initial oxygen and glucose deficits lead to excitotoxicity, disruption of ion homeostasis and calcium dysregulation that affect mitochondria. In a second step, during reperfusion, due to this first energetic failure and the increase in free radicals and reactive oxygen species (ROS) production, cellular swelling takes place and neuronal apoptosis occurs. Indeed, neurons are the most affected by oxygen and glucose deprivation [START_REF] Northington | Early neurodegeneration after hypoxia-ischemia in neonatal rat is necrosis while delayed neuronal death is apoptosis[END_REF][START_REF] Hilario | Cerebral blood flow and morphological changes after hypoxic-ischaemic injury in preterm lambs[END_REF] . Their death results in sensorimotor deficit, mental retardation and learning abnormalities [START_REF] Robertson | Long-term follow-up of term neonates with perinatal asphyxia[END_REF][START_REF] Aridas | Detecting brain injury in neonatal hypoxic ischemic encephalopathy: Closing the gap between experimental and clinical research[END_REF] . The modest response to the unique therapeutic strategy proposed up to now (hypothermia) emphasizes the necessity to find new treatments.

A growing number of studies have shown a neuroprotective role of RSV in a context of neonatal HI. This polyphenol was found to be a potent protective agent after an HI event by reducing infarct size, neuronal loss and, consequently, by preventing cognitive deficits [START_REF] West | Pomegranate polyphenols and resveratrol protect the neonatal brain against hypoxic-ischemic injury[END_REF][START_REF] Karalis | Resveratrol ameliorates hypoxia/ischemia-induced behavioral deficits and brain injury in the neonatal rat brain[END_REF][START_REF] Arteaga | Pretreatment with resveratrol prevents neuronal injury and cognitive deficits induced by perinatal hypoxia-ischemia in rats[END_REF] . However, the neuroprotective effect of RSV was observed after direct intraperitoneal injection in pups that underwent HI, a pro-inflammatory mode of administration. Moreover, in previous studies, RSV was administrated at high pharmacological doses (20-100 mg/kg). In this study, we used, for the first time, a low (nutritional) dose of RSV (0.15 mg/kg/day) and explored the impact of maternal supplementation. This corresponds to an ingested doses of 10.5 mg of RSV or PIC per day for a pregnant woman (weighing 70 kg). RSV is present in various edible plants such as rhubarb, fresh peanut, cocoa beans and in large quantities in grapes, which contain up to 486 mg/kg of RSV [START_REF] Zhao | Composition and antioxidant activity of raisin extracts obtained from various solvents[END_REF] (depending on the specie). Therefore, the doses of RSV administered during our maternal supplementation protocol correspond to the consumption of about 22 g of grapes (about thirty grape berries) per day for a pregnant woman. Similarly, taking into account the PIC content of passion fruit (2.2 mg of PIC per g of raw passion fruit seeds [START_REF] Matsui | Extract of passion fruit (passiflora edulis) seed containing high amounts of piceatannol inhibits melanogenesis and promotes collagen synthesis[END_REF] ) and the weight of the seeds (between 5 and 10 g/fruit) , a passion fruit contains about 10 to 20 mg of PIC. Therefore, the doses of PIC tested in maternal supplementation in the rat correspond to the consumption of a single passion fruit per day for a pregnant woman. Concerning ethanol, the doses used to mimic maternal alcoholism are moderate. Since dams were supplemented with ethanol at a dose of 0.5 g/kg/d, this corresponds to 35 g/d for a pregnant woman (70 kg), which is equivalent to the consumption of 2 glasses of wine (14 cl, 12.5 °). This dose was chosen to correspond to a moderate alcohol consumption and our model reflects the fact that 10% of women still drink alcohol during pregnancy [START_REF] Popova | Estimation of national, regional, and global prevalence of alcohol use during pregnancy and fetal alcohol syndrome: A systematic review and meta-analysis[END_REF] . If the prevalence of alcohol use during pregnancy was estimated by several studies, it is more difficult to get epidemiological data on doses of alcohol intake during this period. However, it has been estimated that among pregnant women drinking a glass of wine daily, 10% will have a child with fetal alcohol syndrome [START_REF] Gop | A synopsis of chlidren's diseases[END_REF] . Therefore, the main aim of the present original nutritional and transgenerational study was to evaluate the effect of a maternal supplementation with RSV or PIC on HI-pup brain damages in a context of moderate maternal alcoholism.

Besides the quantification of lesion volume, the early measurements of ADC reflected cytotoxic edema severity. Indeed, in this case, tissue edema is due to cellular swelling and leads to a decrease in extracellular water movement speed and then, therefore, to a decrease in ADC values. Three major types of cerebral edema have been described: cytotoxic, ionic and vasogenic [START_REF] Berezowski | Endothelial cells and astrocytes: A concerto en duo in ischemic pathophysiology[END_REF] . Cytotoxic edema occurs in the early stage after stroke and is characterized by the swelling of astrocytes and neuronal dendrites [START_REF] Badaut | Aquaporins in cerebrovascular disease: A target for treatment of brain edema?[END_REF][START_REF] Zador | Role of aquaporin-4 in cerebral edema and stroke[END_REF] , due to the energetic failure linked to oxygen and glucose deprivation, which leads to a cellular entry of ions and water. Ionic edema has been described as a continuum between cytotoxic and vasogenic edema and is characterized by the swelling of endothelial cells.

It occurs within 30 minutes after reperfusion [START_REF] Berezowski | Endothelial cells and astrocytes: A concerto en duo in ischemic pathophysiology[END_REF] . The vasogenic edema is linked to the blood-brain barrier disruption and happens at a later stage [START_REF] Badaut | Aquaporins in cerebrovascular disease: A target for treatment of brain edema?[END_REF] . A smaller decrease in ADC values in the cortex and in the striatum was measured in the PIC/Eth group indicating that, in addition to have smaller lesions, pups that underwent HI in this group presented a less severe cytotoxic edema. These data underlined a neuroprotective role of maternal PIC supplementation. This neuroprotective effect of PIC supplementation was also observed at the cellular level by histochemistry since less neuronal death was measured in both cortical and striatal penumbra regions. Surprisingly, maternal Eth consumption also led to a smaller decrease in ADC values but was, however, neither linked to smaller lesion volumes 48 h post-HI nor with neuronal survival determined by the Nissl staining.

This underline the fact that ADC values alone, measured just after the insult, are not sufficient to predict the recovery of the penumbra zone.

Regarding sensorimotor performances, hypoxic-ischemic pups (HIC) performed less well than pups in the sham group for the righting reflex and the negative geotaxis tests. These results are in agreement with previous studies on mice or rats subjected to HI [START_REF] Lubics | Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic-ischemic injury[END_REF][START_REF] Ten | Brain injury and neurofunctional deficit in neonatal mice with hypoxic-ischemic encephalopathy[END_REF][START_REF] Kiss | Effects of perinatal asphyxia on the neurobehavioral and retinal development of newborn rats[END_REF] showing that this event affects the short-term outcome of early reflexes. The deleterious effect of maternal alcoholism could be highlighted by these tests since Eth group pups were not able to improve their performance with time, which was the case for pups in the other HI groups. If maternal supplementation with RSV allowed to partially counteract some deleterious effects of HI, this supplementation was not as neuroprotective as PIC. Indeed, pups in the PIC/Eth group were able to reach the same level of performance as sham pups for these tests, meaning that maternal oral PIC supply improves early deficits after HI injury, even in a context of maternal alcoholism. Pups subjected to neonatal HI show a high capacity of recovery, as it has been suggested by previous studies [START_REF] Lubics | Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic-ischemic injury[END_REF][START_REF] Kiss | Effects of perinatal asphyxia on the neurobehavioral and retinal development of newborn rats[END_REF] and RSV has been shown to decrease the deleterious effects of Eth in hippocampal neurogenesis, in 7-day-old mice [START_REF] Xu | Protective effects of resveratrol on the inhibition of hippocampal neurogenesis induced by ethanol during early postnatal life[END_REF] . Sensorimotor functions were also assessed later in juvenile rats (P20-24) using the adhesive removal test. This test is usually used to detect long-term deficit after stroke in adult rodents [START_REF] Bouet | Predicting sensorimotor and memory deficits after neonatal ischemic stroke with reperfusion in the rat[END_REF][START_REF] Freret | Long-term functional outcome following transient middle cerebral artery occlusion in the rat: Correlation between brain damage and behavioral impairment[END_REF][START_REF] Zhang | A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia[END_REF] and was adapted to juvenile rats by Bouet et al. for assessing somatosensory deficits and asymmetric dysfunctions after neonatal ischemic stroke [START_REF] Bouet | Predicting sensorimotor and memory deficits after neonatal ischemic stroke with reperfusion in the rat[END_REF] . In this test, even if all groups, except the Eth group, improved their performance over time, as a consequence of task learning, HI groups still performed less well than the sham group except the PIC/Eth group which was indistinguishable from the sham group. These results indicate that maternal PIC supplementation counteracted the deleterious effect of the HI event on juvenile somatosensory asymmetries even in a maternal alcoholism context. These data were reinforced by the mNSS results.

It had been reported in a cohort of 40 children that HI can lead to hippocampal damage and, later to an impairment in cognitive memory [START_REF] Cooper | Neonatal hypoxia, hippocampal atrophy, and memory impairment: Evidence of a causal sequence[END_REF] . HI rodents also display short-and long-term memory deficits [START_REF] Arteaga | Pretreatment with resveratrol prevents neuronal injury and cognitive deficits induced by perinatal hypoxia-ischemia in rats[END_REF][START_REF] Balduini | Long-lasting behavioral alterations following a hypoxic/ischemic brain injury in neonatal rats[END_REF][START_REF] Ikeda | Selective and long-term learning impairment following neonatal hypoxic-ischemic brain insult in rats[END_REF][START_REF] Wagner | Delayed postischemic hypothermia improves long-term behavioral outcome after cerebral hypoxia-ischemia in neonatal rats[END_REF] . Long-term memory was assessed by the novel object recognition test, in which maternal PIC supplementation was able to revert the deleterious effect of HI and maternal alcoholism (same discrimination index for PIC/Eth and sham groups). These data show a neuroprotective role of maternal PIC supplementation on hippocampal-dependent long-term memory of pups. These results are in agreement with the study of Arteaga et al. [START_REF] Arteaga | Pretreatment with resveratrol prevents neuronal injury and cognitive deficits induced by perinatal hypoxia-ischemia in rats[END_REF] , which demonstrated that intraperitoneal injection of RSV improves the long-lasting cognitive deficits induced by HI (a study not in the context of alcoholism however). They proposed that the RSV protective effect could be due to the protection and preservation of neocortical and subcortical brain areas (sensorimotor cortex, hippocampus and striatum) and in particular, of neuronal networks responsible for learning and memory [START_REF] Arteaga | Pretreatment with resveratrol prevents neuronal injury and cognitive deficits induced by perinatal hypoxia-ischemia in rats[END_REF] . The authors postulated in this article that the neuroprotective role of RSV could be due to its antioxidant effect. Pups in the Eth group presented the lowest discrimination index, indicating that long-term memory was impaired, even in a context of moderate maternal alcohol consumption. Such an impairment was also observed in a context of mild focal traumatic brain injury coupled with alcohol exposition (sub-chronic intermittent alcohol vapour (95% ethanol 14h on/10h off) [START_REF] Teng | Alcohol exposure after mild focal traumatic brain injury impairs neurological recovery and exacerbates localized neuroinflammation[END_REF] . It could be noted also that pups in the RSV/Eth group tended to present intermediate discrimination index values, suggesting that RSV could prevent, but only partially, hippocampal-dependant long-term memory deficits induced by ethanol consumption.

Many studies have focused on the antioxidant properties of RSV. However, these properties are observed only at high doses. In the context of HI, RSV neuroprotection could be multimodal. In addition to SIRT1 activation [START_REF] Yang | New role of silent information regulator 1 in cerebral ischemia[END_REF] , RSV could also modulate metabolism (Fig. 4). For example, HI leads to a decrease in astrocytic glutamate uptake [START_REF] Morken | Altered astrocyteneuronal interactions after hypoxia-ischemia in the neonatal brain in female and male rats[END_REF] , which results in glutamate excitotoxicity that could be reversed by RSV i.p. injection in a rat model of transient forebrain ischemia [START_REF] Girbovan | Resveratrol downregulates type-1 glutamate transporter expression and microglia activation in the hippocampus following cerebral ischemia reperfusion in rats[END_REF] . The same was observed in a rat hippocampal slice model of H2O2-induced oxidative injury [START_REF] De Almeida | Resveratrol protects against oxidative injury induced by h2o2 in acute hippocampal slice preparations from wistar rats[END_REF] . In another study, in rat hippocampal slices exposed to glutamate, the astrocytic recycling of glutamate to glutamine was increased after RSV exposure (100 µM). Similar data were also obtained in primary astrocyte cultures of P1-pups in presence of RSV (10 µM) [START_REF] Bellaver | Resveratrol increases antioxidant defenses and decreases proinflammatory cytokines in hippocampal astrocyte cultures from newborn, adult and aged wistar rats[END_REF] . Moreover, in primary cultures of mouse cortical astrocytes, it has been shown that the uptake of glutamate induces an increase in astrocytic production of lactate via the stimulation of glycolysis [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: A mechanism coupling neuronal activity to glucose utilization[END_REF] . Interestingly, RSV has been shown to increase glycolysis in the liver [START_REF] Beauvieux | Resveratrol mainly stimulates the glycolytic atp synthesis flux and not the mitochondrial one: A saturation transfer nmr study in perfused and isolated rat liver[END_REF] , but also in the brain, leading to a neuroprotective effect in a mouse model of middle cerebral artery occlusion [START_REF] Koronowski | Neuronal sirt1 (silent information regulator 2 homologue 1) regulates glycolysis and mediates resveratrolinduced ischemic tolerance[END_REF] . In parallel, in rat hippocampal slices, RSV (100 µM) led to an increase in lactate release [START_REF] Quincozes-Santos | Oxidative stress mediated by nmda, ampa/ka channels in acute hippocampal slices: Neuroprotective effect of resveratrol[END_REF] . Although lactate has long been considered as a metabolic waste product, it is now more and more considered as a substantial brain energetic fuel.

In 1994, Pellerin and Magistretti proposed a metabolic exchange between astrocytes and neurons in which blood glucose enters first the astrocytes, which are more glycolytic cells [START_REF] Bouzier-Sore | Unraveling the complex metabolic nature of astrocytes[END_REF] , where it is converted into lactate. This lactate is then transferred to neurons where it is converted back to pyruvate for its oxidative use [START_REF] Bouzier-Sore | Lactate is a preferential oxidative energy substrate over glucose for neurons in culture[END_REF] , an intercellular exchange called the astrocyte-neuron lactate shuttle (ANLS) [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: A mechanism coupling neuronal activity to glucose utilization[END_REF] . In the context of HI, providing lactate as an energetic substrate for neurons could spare glucose for its metabolic use through the pentose phosphate pathway (PPP). Indeed, this pathway is mandatory to maintain high levels of reduced glutathione, a metabolite necessary to fight against ROS. Glutathione levels were measured in neuronal stem cells treated with RSV (5 µM) following oxygen-glucose deprivation/reoxygenation 62 as well as in rat hippocampal slices treated with RSV (100 µM), in a context of H2O2 oxidative stress injury [START_REF] De Almeida | Resveratrol protects against oxidative injury induced by h2o2 in acute hippocampal slice preparations from wistar rats[END_REF] . The content of glutathione was increased by 20% and 80%, respectively. In astrocytes, RSV (10 mM) was able to decrease intracellular ROS levels induced by H2O2 oxidative stress [START_REF] Daverey | Pre and post treatment with curcumin and resveratrol protects astrocytes after oxidative stress[END_REF] . By increasing glycolysis, and therefore lactate, in astrocytes (which could then promote glucose use via PPP in neurons and enhanced their reduced glutathione levels), RSV may prevent neuronal death and reduce brain damages leading to motor and cognitive impairments.

In this study, RSV was not able to fully protect against HI lesions in a context of moderate alcohol maternal intake. However, PIC supplementation, even combined with a moderate dose of ethanol, allowed pups to recover their sensorimotor and cognitive functions after an HI event. PIC differs from its analogue RSV by only one hydroxyl group. This confers to PIC a higher bioavailability compared to RSV [START_REF] Kukreja | Therapeutic role of resveratrol and piceatannol in disease prevention[END_REF] , which could explain the higher neuroprotective effect of PIC compared to RSV. Contrary to a comprehensive literature on RSV, few publications report on the effects of PIC; our results argue in favor of the need to continue studies on this molecule.

Taken altogether, our results support that in a context of neonatal HI and moderate maternal alcohol consumption, PIC, at a dose equivalent in humans to 1 passion fruit / day, could partially reverse the anatomical brain lesions and, more importantly, could totally counteract the sensorimotor and long-term memory impairments. Based on these results, maternal supplementation with PIC may be considered as a preventive nutritional approach in the context of maternal alcoholism to limit further possible brain damages if a neonatal HI event might occur.

Experimental procedures

Study design

The aim of the present work was to study the effect of maternal nutritional supplementation with polyphenols on offspring brain damages induced by HI, in a moderate maternal alcoholism context. Scheme of the experimental design is presented in Fig. 5. All animal procedures were conducted in accordance with the Animal Experimentation Guidelines of the European Communities Council Directive of November 24, 1986 (86/609/EEC). Protocols met the ethical guidelines of the French Ministry of Agriculture and Forests, and were approved by the ethical committees (N° authorization 9476). Sample size was determined based on a previous study 64 .

Animals

Pregnant Wistar RJ-HAN females (Janvier Laboratories, Le Genest Saint Isle, France) were received one week before delivery and were kept on a 12:12 h light:dark cycle with water and food (A03) ad libitum.

Model of hypoxic-ischemic brain injury

Cerebral HI was performed as described previously [START_REF] Rice | The influence of immaturity on hypoxic-ischemic brain damage in the rat[END_REF][START_REF] Dumont | Maternal consumption of piceatannol: A nutritional neuroprotective strategy against hypoxia-ischemia in rat neonates[END_REF][START_REF] Brissaud | Short-term effect of erythropoietin on brain lesions and aquaporin-4 expression in a hypoxic-ischemic neonatal rat model assessed by magnetic resonance diffusion weighted imaging and immunohistochemistry[END_REF] . Briefly, P7-pups of either sex, weighing 14-20 g, were anaesthetized with isoflurane (induction: 4%; maintenance: 1.5%) and a midline neck incision was made under local anesthesia (lidocaine 0.5%). The left common carotid artery was exposed and permanently ligated (7-0 silk thread). After surgery, animals were allowed to recover (30 min) on a heated mattress to maintain body temperature at 36 ± 1°C. Pups were then placed for 2 h in a humidified hypoxic chamber (8% oxygen, 92% nitrogen -33°C) (Intensive Care Unit Warmer, Harvard Apparatus, Les Ulis, France) to maintain a constant thermal environment (rectal temperature: 36 ± 1°C). During hypoxia, sham-operated pups were kept separated from the dam in a heated atmosphere (33°C). The first diffusion MRI was performed 3 h after carotid ligation, then after 48 h (P9) and 23 days (P30).

Experimental groups

Five experimental groups were considered. The different nutritional supplementations were added in the maternal drinking water 7 th day before delivery until the 9 th day post-delivery.

Maternal drinking water was supplemented with Eth (Sigma-Aldrich, France; 0.5 g/kg/dequivalent to the consumption of 2 glasses of wine (14 cl, 12.5 °) for a pregnant woman) for Eth group (n=19), Eth (0.5 g/kg/d) and RSV (Sigma-Aldrich, France; 0.15 mg/kg/d) for RSV/Eth group (n = 10) and Eth (0.5 g/kg/d) and PIC (Tokyo Chemical Industry Co, France; 0.15 mg/kg/d) for PIC/Eth group (n = 12). For sham (n = 8) and control groups (HIC group, n = 17), 4 µl of absolute Eth were added into the rat water bottle (corresponding to the Eth volume added during polyphenols supplementation (final Eth concentration: 0.002%) since polyphenol solutions were prepared in absolute ethanol).

Additional controls were also performed to ensure that maternal RSV or PIC supplementation alone was not deleterious for pups (MRI and behavioural parameters, data not shown). For these two supplementary groups, dams were supplemented with RSV (n = 8) or PIC (n = 8) (0.15 mg/kg/d), respectively, during the same period.

Acquisition of in vivo longitudinal MRI.

In vivo MRI was performed at P7 (3 h after carotid ligation), P9 and P30 on a horizontal 4.7T Biospec 47/50 system (Bruker, Ettlingen, Germany) equipped with a 6-cm BG6 gradient system (1000 mT/m, Δ=20ms, δ=4ms). For MRI, pups were anesthetized with isoflurane 4% for induction and 1.5 ± 1% for maintenance. Respiration was monitored by a ventral pressure sensor. Pup body temperature was maintained at 36 ± 1°C during acquisitions with a water-heated MRI bed. Anatomical T2-weighted images of the brain were obtained using a Rapid Acquisition with Relaxation Enhancement (RARE) sequence with 20 axial slices, 0.7 mm thick, echo time (TE) = 50 ms, repetition time (TR) = 3000 ms (total duration 4 min 48 s). Brain lesions were assessed by Diffusion Weighted Imaging (DWI: 20 axial slices of 0.7 mm, 30 directions, b-value 1347 s/mm 2 , TE 24 ms, TR 2 s, total duration 17 min 04 s). Measurements were performed with Paravision 6.0.1 software (Bruker BioSpin, Karlsruhe, Germany).

MRI Analysis

The lesion volumes were measured on the ADC maps on 20 successive slices at P7, P9 and on T2 weighted images at P30 and are presented as a % of the total brain volume. For each rat, two regionof-interest (ROI) were manually drawn, on each slice (n = 20), the first one that encompasses the injured area and the second one to determine the global brain area. Edema severity was evaluated by the measurement of ADC values at P7. ADC maps were computed using the Paravision 6.0.1 software diffusion tensor calculation module. ADC values were measured inside the lesion area.

ROIs were manually delineated in each injured structure (cortex, hippocampus, striatum). Analyses were performed blind by 2 separate operators (intra-operator variability < 1%; inter-operator variability < 5%). Pups were placed on their back and the time they took to turn over their four paws was recorded.

Each pup was given three trials a day and the mean time to perform the test was calculated.

Adhesive removal test (from P20 to P24)

Somatosensory deficits and asymmetric dysfunctions were evaluated using the adhesive removal test [START_REF] Bouet | Predicting sensorimotor and memory deficits after neonatal ischemic stroke with reperfusion in the rat[END_REF][START_REF] Chen | Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[END_REF] . After an habituation period (60 s) in the open field (20 cm x 30 cm), two adhesive tapes (0.5 cm x 0.5 cm) were used as bilateral tactile stimuli occupying the distal-radial region on the wrist of each forelimb. Time to contact (control, no significant difference between groups, data not shown) and to remove the adhesive were measured during one trial per day (120 s maximum).

Modified neurological severity score (mNSS) (P24)

Neurological functions were assessed using the mNSS [START_REF] Chen | Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[END_REF] , which is composed of motor (muscle status), sensory (tactile and proprioceptive), beam balance and reflexes (pinna, corneal and startle) tests. Each rat was graded on the mNSS scale (<1: no impairment -1-6: middle impairment -7-12: moderate impairment -13-18: severe impairment).

Novel object recognition (P45) 31

Hippocampal-dependent long-term memory of pups was assessed using the novel object recognition test, as described previously [START_REF] Dumont | Maternal consumption of piceatannol: A nutritional neuroprotective strategy against hypoxia-ischemia in rat neonates[END_REF] . Briefly, after an habituation period (10 min) in the open field (50 cm x 50 cm) on day 1, pups were further allowed to freely explore two identical objects placed in opposite corners of the arena for 10 min during the following two days. On the day after (day 4), one of the previous object was replaced by a different one and pups were allowed to freely explore the objects for 10 min. Duration of object exploration was measured. The discrimination index was calculated as followed: ([time spent on new object -time spent on familiar object]/[time spent on new object + time spent on familiar object]).

Nissl staining

In addition to MRI and behavioral tests, Nissl staining was performed on P9-pups brain sections, as described previously [START_REF] Dumont | Maternal consumption of piceatannol: A nutritional neuroprotective strategy against hypoxia-ischemia in rat neonates[END_REF] , to highlight neuronal death in the penumbra region. Briefly, pups were anesthetized (isofluorane 4.5%) and were perfused transcardiacally first with PBS (1X) then with paraformaldehyde (PFA 4%) for 10 min. Removed brain were post-fixed in PFA over night, cryoprotected by sucrose immersion and then frozen in liquid N2 vapor. The 16µm-thick brain sections were stained with 0.5% cresyl violet (Sigma Aldrich, France) for 5 min and washed in distilled water for 10 s.

Statistical Analysis

Statistical analysis and graphs were performed using GraphPad Prism 7.00 software. All data were expressed as means ± standard error of the means (SEM). Statistical significance of the differences between multiple groups was determined using One-Way ANOVA followed by a Fisher's LSD post hoc test. Statistical significance was defined as p < 0.05. Number of pups was stated with each result along with the statistical test used.

Sources of Funding

This work was supported by grants (2018/01) from the Fondation de Recherche en Alcoologie (FRA) from the French State in the context of the "Investments for the future" Programme IdEx (ANR-10-IDEX) and the LabEx TRAIL (ANR-10-LABX-57) and a French-Swiss ANR-FNS grant (ANR-15-CE37-0012).

Conflict of Interest

Authors declare that they have no conflict of interest.

4. 7 .

 7 Behavioral study 4.7.1. Righting reflex (P8, P10, P12)

Fig. 1

 1 Fig. 1 Early impact of maternal nutritional supplementation on neonate brain injuries

Fig. 2

 2 Fig. 2 Long term impact of maternal nutritional supplementation on neonate brain injuries

Fig. 3

 3 Fig. 3 Early and long-term effect maternal supplementation on sensorimotor and cognitive

Fig. 4 Fig. 5

 45 Fig. 4 Schematic representation of RSV effects on brain metabolism.

Statement of financial support: This work was supported by grants (2018/01) from the Fondation de Recherche en Alcoologie (FRA) from the French Government in the context of the "Investments for the future" Programme IdEx (ANR-10-IDEX) and the LabEx TRAIL (ANR-10-LABX-57) and a French-Swiss ANR-FNS grant (ANR-15-CE37-0012). declare no conflict of interest.


Adrien Boes

Jean-Michel Brunel

Adeline Derouaux

Frédéric Kerff

Ahmed Bouhss

Thierry Touze

Eefjan Breukink

Mohammed Terrak
email: mterrak@uliege.be

Squalamine and aminosterol mimics inhibit the peptidoglycan glycosyltransferase activity of PBP1b

Keywords: Peptidoglycan, Lipid II, PBP1b, squalamine, cationic aminosterols

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional PBPs of class A, such as E. coli PBP1b, belong to the GTase 51 family. They play essential role in PG synthesis and their specific inhibition by moenomycin lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds presented an unexpected additional mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay suggesting that they act as competitive inhibitors.

Introduction

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target; it surrounds the cytoplasmic membrane, determines the cell shape and protects the cell from lysis. The PG structure consists of glycan strands made of alternating β-1,4-linked Nacetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues cross-linked by peptides [START_REF] Vollmer | Peptidoglycan structure and architecture[END_REF]. It is assembled using the lipid II precursor (undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc) by the glycosyltransferases (GTases) activities of the class A penicillin-binding proteins (aPBPs) and SEDS proteins and cross-linked by the transpeptidases (TPases) activities of aPBPs and class B PBPs (bPBPs) [START_REF] Sauvage | The penicillinbinding proteins: structure and role in peptidoglycan biosynthesis[END_REF][START_REF] Meeske | SEDS proteins are a widespread family of bacterial cell wall polymerases[END_REF][START_REF] Taguchi | FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein[END_REF]. The inhibition of each one of those activities lead to cell lysis and the death of the bacteria [START_REF] Cho | Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously[END_REF].

The glycosyltransferases (GTase) domain of bifunction PBPs of class A, such as E. coli PBP1b, belong to the GTase 51 family. The enzymatic cavity of these proteins can be divided into two substrate binding sites: a donor site for the lipid-bound growing glycan chain, and an acceptor site for lipid II [START_REF] Sung | Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli[END_REF]. Moenomycin A is the only known natural product that specifically binds to the GTase 51 donor site and competitively inhibits PG synthesis [START_REF] Welzel | Syntheses around the transglycosylation step in peptidoglycan biosynthesis[END_REF]. It is a potent antibiotic that is not used in human therapy but was used in animal feedstock for decades without reported resistance [START_REF] Butaye | Influence of different medium components on the in vitro activity of the growth-promoting antibiotic flavomycin against enterococci[END_REF].

Squalamine is a cationic aminosterol antibiotic, isolated from the dogfish shark (Squalus acanthias) [START_REF] Moore | An aminosterol antibiotic from the shark[END_REF], exhibiting a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria and potentiate the activity of several antibiotic classes at subinhibitory concentrations, against both susceptible and resistant bacteria [START_REF] Douafer | Antibiotic Adjuvants: Make Antibiotics Great Again![END_REF]. Its structure is composed of a steroid linked to a polyamino spermidine moiety, which taken independently exhibit considerably low antibiotic activity and are both required for the observed antimicrobial activity of squalamine [START_REF] Moore | An aminosterol antibiotic from the shark[END_REF].

Since more than one decade, numerous aminosterol derivatives of squalamine have been synthesized and characterized [START_REF] Salmi | Antimicrobial activities of 3-amino-and polyaminosterol analogues of squalamine and trodusquemine[END_REF][START_REF] Kikuchi | Antimicrobial activities of squalamine mimics[END_REF][START_REF] Djouhri-Bouktab | Synthesis of new 3,20bispolyaminosteroid squalamine analogues and evaluation of their antimicrobial activities[END_REF][START_REF] Choucair | Synthesis of 7α-and 7βspermidinylcholesterol, squalamine analogues[END_REF][START_REF] Brycki | Quaternary alkylammonium conjugates of steroids: Synthesis, molecular structure, and biological studies[END_REF]. Their mode of action was proposed to act by disrupting the outer membrane integrity of Gram-negative bacteria and by depolarizing the cytoplasmic membrane of Gram-positive bacteria [START_REF] Savage | Antibacterial properties of cationic steroid antibiotics[END_REF][START_REF] Di Pasquale | Biophysical studies of the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic membranes: importance of the distribution coefficient in membrane selectivity[END_REF]. Some of them display good membrane selectivity with high activity on bacterial membrane and low hemolytic activity, which make them promising candidate as new antibiotics or as adjuvants to sensitize resistant bacteria to the wellknown antibiotics [START_REF] Savage | Antibacterial properties of cationic steroid antibiotics[END_REF][START_REF] Di Pasquale | Biophysical studies of the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic membranes: importance of the distribution coefficient in membrane selectivity[END_REF].

Herein, we will demonstrate that squalamine and other aminosterol mimics present an unexpected additional mode of action, consisting of the inhibition of a bacterial peptidoglycan polymerase. By using in vitro activity assays based on the processing of lipid II substrate into peptidoglycan polymers by E. coli PBP1b, we found that squalamine and six other aminosterol analogs inhibit the GTase activity of this enzyme. In addition, the selected compounds were able to displace the lipid II from the active site in a fluorescence anisotropy assay indicating that they act as competitive inhibitors.

Results

Squalamine inhibits the GTase activity of PBP1b

The crystal structure of the glycosyltransferase domain of the class A PBP1a from Aquifex aeolicus presents a steroidal CHAPS detergent molecule bound to a hydrophobic patch that lead to the active site [START_REF] Yuan | Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis[END_REF]. Its side chain was suggested to mimic and partly overlap with diphospholipid chain of the lipid II substrate at the acceptor site. We noticed that the aminosterol antibiotic squalamine and CHAPS have some structural feature in common, consisting essentially of a sterane core (Fig. 2). We were curious to know if squalamine could affect the peptidoglycan polymerase activity of E. coli PBP1b. Surprisingly, by using radioactive lipid II as substrate in an endpoint TLC assay we found that squalamine was able to almost completely inhibit the glycosyltransferase activity of PBP1b (1.1 ± 0.6 % residual activity (RA) at 0.8 mM) (Fig. 1A). Variable concentrations of squalamine were then tested using both an endpoint TLC assay and a continuous fluorescence assay that monitor the consumption of dansyl-lipid II substrate over time. The inhibition result shows concentrationsdependent profile and the efficacy of squalamine was rather modest in the high µmolar range (IC50 value of 291 ± 26 µM) (Fig. 1B, Table 1). When the zwitterionic CHAPS detergent was tested using the same assay at high concentrations and below the critical micelle concentration (CMC 6-10 mM) value, no inhibition of PBP1b GTase activity was observed up to 5 mM (Fig. 1C). In addition, steroid progesterone11 or octanediamine 12tested alone do not inhibit GTase activity of PBP1b (Fig. 2, Table 1).

Inhibition of the GTase activity of PBP1b by aminosterol mimics of squalamine

Based on these encouraging results we have analyzed twelve additional aminosterol compounds with one or more substitutions at C-3, C-6, C-7 and C-17 positions of the sterane rings. To determine the relative activity of the compounds, they were tested at 400 and 800 µM and ranked according to their effect on the residual activity (RA) of the enzyme. Six compounds were found to inhibit the glycosyltransferase activity of PBP1b. Thus, , the residual GTase activity of PBP1b ranged from 0.3 to 8.5 % and from 1.2 to 39 % by using compound concentrations of 400 and 800 µM, respectively (Fig. 2, Table 1). The active compounds were investigated further to determine the IC50 values (Table 1) and the best derivatives 6 and 2 were found about three time more efficient that squalamine with an IC50 values of 111 ± 6 µM and 114 ± 6 µM, respectively. Interestingly, compound 9, a close analog of compound 2 which is a stereoisomer at position C-7, was almost inactive (RA 98 % at 800 µM). A second observation that can be made is that the active compounds (except for squalamine) possess nonpolar moiety in C-17. In contrast, the presence of polar groups at this position tends to abolish the activity of the compounds (e.g amino group in 10, 13, 14, 15). Altogether; these results indicate that the observed inhibitions of the GTase activity of PBP1b by squalamine and analogs are specific and exclude the possibility of promiscuous effect of these series of compounds.

Mode of action of aminosterol compounds

The inhibition of the GTase activity can be achieved either by compounds that bind to the active site of the enzyme (e.g. moenomycin A) or through binding to the lipid II substrate (e.g. nisin, ramoplanin). To distinguish between these two mechanisms, we further characterized the mode of action of squalamine, the best inhibitory compound (6) and an inactive compound (15) by studying their interaction with PBP1b and lipid II, using a recently developed fluorescence anisotropy assay based on a fluorescent lipid II as a probe [START_REF] Boes | Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ[END_REF]. The assay was previously validated for direct interaction of the probe with PBP1b and with the antibiotics nisin and ramoplanin (FA increase upon binding), as well as the ability of unlabeled-lipid II, nisin or ramoplanin to displace the probe form the enzyme active site (FA decrease in the presence of competitive compound) [START_REF] Boes | Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ[END_REF]. While no significant interaction was observed between lipid II and squalamine or compounds 6 and 15, the active compounds were able to compete with lipid II for binding to PBP1b, whereas the inactive compound 15 had no effect. These results clearly indicate that the active aminosterol compounds directly interact with the glycosyltransferase active site of PBP1b (Fig. 3). All together the data reveal that squalamine and active analogs have multiple antibacterial mode of action, in addition to their potent membranes damaging activities, they also have modest cell wall synthesis inhibition activity.

Discussion

The prevalence of antibacterial resistance is increasing and new solutions should be developed urgently to deal with the resistant pathogens. The GTase activity of bifunctional PBPs is essential for bacterial viability and constitute a proven antibacterial target [START_REF] Derouaux | Peptidoglycan glycosyltransferase substrate mimics as templates for the design of new antibacterial drugs[END_REF][START_REF] Sauvage | Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials[END_REF]. However, only one natural product antibiotic, moenomycin, that specifically bind to the active site of these enzymes has been discovered more than 50 years ago [START_REF] Welzel | Syntheses around the transglycosylation step in peptidoglycan biosynthesis[END_REF][START_REF] Ostash | Moenomycin family antibiotics: chemical synthesis, biosynthesis, and biological activity[END_REF], and therefore finding a new chemical scaffold able to bind and inhibits this class of enzymes is of great importance. In this regard, the discovery of the natural product squalamine and synthetic analogues that inhibit the GTase activity of the model class A PBP1b enzyme open new perspective for the development of new class of GTase inhibitors. The identification of aminosterol derivatives with improved activity against the GTase, while maintaining some specific bacterial membranes damaging activity, could result in potent antibacterial agents, particularly against resistant Gram-negative bacteria.

Interestingly, FA experiments indicate that these compounds are competitive inhibitors able to displace the lipid II substrate from the GTase active site, but in contrast to moenomycin which bind only to the donor site and shows an increase in FA upon cooperative binding with lipid II substrate to PBP1b [START_REF] Boes | Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ[END_REF][START_REF] Bury | Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase[END_REF], the titration of the PBP1b-probe complex by aminosterol compounds induce a decrease in FA signal, similar to that observed in the presence of unlabeled lipid II, indicating that they prevent lipid II probe binding to both donor and acceptor sites.

The observation that the presence of amino group at the C17 substitution abolish the activity of the compounds (10, 13, 14, 15), may indicate that this part of the molecules interact with the positively charged residues of the active site, potential candidates would be the positive pocket in the donor site formed by residues (K274, R282, R286, R287) [START_REF] Sung | Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli[END_REF][START_REF] Terrak | Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli[END_REF]. Positioning of a CHAPS molecule into the active site of PBP1b by homology to PBP1a-CHAPS structure [START_REF] Yuan | Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis[END_REF] suggest that this may be the case (Fig 4).

Moenomycin has an excellent antibacterial potency and no reported resistance despite extensive use in animal feeds, but it is not used in human therapy because of it its undesirable physical properties that result in poor bioavailability and long serum half-life, probably due to its lipidic C25 moenocinol moiety [START_REF] Butaye | Influence of different medium components on the in vitro activity of the growth-promoting antibiotic flavomycin against enterococci[END_REF][START_REF] Pfaller | Flavophospholipol use in animals: positive implications for antimicrobial resistance based on its microbiologic properties[END_REF][START_REF] Hentschel | Staphylococcus aureus in poultry--biochemical characteristics, antibiotic resistance and phage pattern (author's transl)[END_REF]. This lipid tail is necessary for moenomycin activity and its replacement by an alternative one could improve the properties of the resulting molecule. The C25 lipid tail of moenomycin was not resolved in any crystal structure of GTase-moenomycin complexes, indicating that it is not bound in an ordered conformation [START_REF] Sung | Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli[END_REF][START_REF] King | Structural insights into inhibition of Escherichia coli penicillin-binding protein 1B[END_REF]. In contrast, CHAPS was well defined in the structure of the GTase domain of PBP1a from A. aeolicus [START_REF] Yuan | Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis[END_REF] suggesting that binding of the cyclic steroid-substituted compounds would provide a defined structure and facilitate structure-based optimization of compounds.

Most of GTase inhibitors (moenomycin and synthetics compounds) are inactive against Gramnegative bacteria [START_REF] Sauvage | Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials[END_REF]. On the other hand, aminosterols have the capacity to cause lesions in the outer membrane and either kill or sensitize Gram-negative resistant strains to common antibiotics. Therefore, it is tempting to speculate that aminosterol compounds may be useful substitute to the moenocinol moiety of the moenomycin or analogs (or to the lipid tail of lipid II analogs [START_REF] Dumbre | Synthesis of modified peptidoglycan precursor analogues for the inhibition of glycosyltransferase[END_REF]) to generate useful new hybrid compounds, consisting of the carbohydrate and phosphoglycerate moieties or the minimal moenomycin pharmacophore (disaccharide (EF)phosphoglycerate [START_REF] Sauvage | Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials[END_REF]) and an aminosterol tail (Fig. 4). Such compounds would have two functions: i) to replace the lipid tail of moenomycin derivatives while maintaining their binding to the GTase active site; and ii) to allow these compounds to cross the outer membrane (via the polyamine cationic moiety) and gain effectiveness against Gram-negative pathogens.

Materiel and Methods

Reagents and protein. Labelled lipid II [N-acetylglucosaminyl-N-acetylmuramoyl (L-Ala-γ-

DGlu-(L)-Lys-(L)-D-Ala-D-Ala)-pyrophosphate-undecaprenol] variants were prepared as previously described: Dansyl-lipid II [START_REF] Breukink | Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes[END_REF], NBD-lipid II [START_REF] Van Dam | Transmembrane transport of peptidoglycan precursors across model and bacterial membranes[END_REF], [ 14 C]-Lipid II (0.06 µCi nmol -1 ) [START_REF] Bouhss | Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis[END_REF].

PBP1bγ(M46-N844) was purified as previously described [START_REF] Terrak | The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli[END_REF].

Compounds synthesis.

The procedures for the synthesis of compounds are described in the supplementary material section. All the solvents were purified according to reported procedures, and the reagents used were commercially available. Methanol, ethyl acetate and dichloromethane were purchased from SDS and used without further purification. Column chromatography was performed on SDS silica gel (70-230 mesh). 1 H NMR and 13 C NMR spectra were recorded in MeOD on a Bruker AC 300 spectrometer working at 300 MHz and 75 MHz, respectively (the usual abbreviations are used: s: singlet, d: doublet, t: triplet, q: quadruplet, m: multiplet). Tetramethylsilane was used as the internal standard. All chemical shifts are given in ppm. Mass spectroscopy analysis have been performed by the Spectropole (Analytical Laboratory) of Aix-Marseille Université (Marseille). The purity of the compounds was checked by analytical HPLC (C18 column, eluent CH3CN-water-TFA (90:10:0.025, v/v/v), 0.5-1 mL/Min) with PDA detector spanning from 210 nm to 310 nm. All compounds possessed purity above 95%, as determined by analytical LCMS Agilent.

Octanediamine 11 and progesterone 12 were purchased from Sigma-aldrich (Saint Quentin Falavier, France). Squalamine 1 was prepared according to reported procedures [START_REF] Zhang | Synthesis of Squalamine Utilizing a Readily Accessible Spermidine Equivalent[END_REF][START_REF] Brunel | Recent Advances in the Synthesis of Spermine and Spermidine Analogs of the Shark Aminosterol Squalamine[END_REF].

Derivatives 2, 3 and 9 were prepared according [START_REF] Loncle | Antimicrobial Activities of 7-Aminosterol Squalamine Analogues[END_REF]. Derivative 4 was prepared according [START_REF] Loncle | Synthesis of new 7-aminosterol squalamine analogues with high antimicrobial activities through a stereoselective titanium reductive amination reaction[END_REF].

Derivatives 10, 13 and 14 were prepared according [START_REF] Djouhri-Bouktab | Synthesis of new 3,20bispolyaminosteroid squalamine analogues and evaluation of their antimicrobial activities[END_REF]. Claramine A1 8 was prepared according [START_REF] Blanchet | Claramines: A New Class Of Broad-Spectrum Antimicrobial Agents With Bimodal Activity[END_REF].

General procedure for the titanium-mediated reductive amination reaction:

6-(spermine)-cholestan-3-ol 6:

A mixture of 6-ketocholestanol (157 mg, 0.39 mmol), titanium(IV) isopropoxide (573 µL, 2.02 mmol) and spermine (202 mg, 1 mmol) in absolute methanol (5 mL) was stirred under argon at room temperature for 12 h. Sodium borohydride (38 mg, 1 mmol) was then added at -78 ºC and the resulting mixture was stirred for an additional 2 hours. The reaction was then quenched by adding water (1 mL) and stirring was maintained at room temperature for 20 minutes. The resulting inorganic precipitate was filtered off over a pad of Celite and washed with methanol and ethylacetate. The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to afford the expected crude amino derivative which was purified by flash chromatography affording the expected amino derivative. Purification by column chromatography (silica gel; CH2Cl2/ MeOH/ NH4OH(32%), 7:3:1) afforded a pale yellow solid in 45% yield; this compound can be converted subsequently into its hydrochloride salt as white solid -1 H NMR (300 MHz, CD3OD) : δ = 0.67-0.76 (m, 4H), 0.90-1.17 (m, 24H), 1.20-1.46 (m, 7H), 1.47-1.92 (m, 20H), 2.03-2.07 (m, 1H), 2.50-2.54 (m, 1H), 2.63-2.73 (m, 13H), 3.56-3.63 (m, 1H) - 13 C NMR (75 MHz, CD3OD) : δ = 71.50, 58.96, 56. 27, 56.05, 54.78, 49.98, 49.21, 47.99, 47.84, 47.34, 42.62, 40.47, 39.94, 39.49, 39.06, 36.44, 36.14, 35.86, 35.77, 35.63, 33.58, 31.61, 30.45, 28.18, 27.97, 24.37, 23.78, 22.78

6-(1,4-diaminobutane)-cholestan-3-ol 5:

Purification by column chromatography (silica gel; CH2Cl2/ MeOH/ NH4OH(32%), 7:3:1) afforded a pale yellow solid in 73% yield; -1 H NMR (300 MHz, MeOD):  = 0.66-3.57 (m, 58H) - 13

N1,N1'-((3S,7R,10R,13R,17R)-10,13-dimethyl-17-((R)-5-methylhexan-2-yl)tetradecahydro-1H-cyclopenta[a]phenanthrene-3,7-diyl)bis(pentane-1,5-diamine) 7

Synthesis according a similar protocol than for derivative 5 but using 3,7-diketocholestene and 1,5-diaminopentane as starting materials. Purification by column chromatography (silica gel; CH2Cl2/ MeOH/ NH4OH(32%), 7:3:1) afforded a pale yellow solid in 48% yield; -1 H NMR (300 MHz, MeOD):  = 0.68-0.70 (m, 3H), 0.83-0.87 ( m, 7H), 0.91-0.93 (m, 3H), 1.01-1.03 (m, 3H), 1.06-1.08 (m, 2H), 1.09-1.18 (m, 4 H), 1. [START_REF] Yuan | Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis[END_REF]

6H-cyclopenta[a]phenanthren-3-ol 15

Synthesis according a similar protocol than for derivative 5 but using oestrone and 1,4butanediamine as starting materials. Purification by column chromatography (silica gel; CH2Cl2/ MeOH/ NH4OH(32%), 7:3:1) afforded a pale yellow solid in 37% yield; -1 H NMR (300 MHz, MeOD):  = 0.77-1.58 (m, 17H), 2.02-2.50 (m, 3H), 2.66-2.74 (m, 5H), 3.13-3.30 (m, 3H), 3.43-3.63 (m, 2H), 6.50-7.08 (m, 3H), 8.06 (s, 1H). - 13 C NMR (75 MHz, MeOD):  = 156.55, 139. 17, 132.80, 127.63, 116.65, 114.32, 70.45, 53.94, 45.69, 44.57, 42.29, 40.75, 40.60, 36.24, 31.15, 30.62, 29.13, 28.10, 24.87, 23.05, 20.46, 14.74, 12.77, 12.69

Glycosyltransferase assays

Continuous fluorescence assay. The GTase activity of PBP1b was monitored by a continuous fluorescent assay using dansyl lipid II as substrate. It was performed in a medium binding black 96-well microplate at 30°C (Greiner Bio One) as described [START_REF] Offant | Optimization of conditions for the glycosyltransferase activity of penicillinbinding protein 1a from Thermotoga maritima[END_REF][START_REF] Schwartz | A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP1b and development of a continuous fluorescence assay[END_REF] Glycosyltransferase activity assay using TLC. The end-point assay, based on radiolabeled lipid II ([ 14 C]lipid II), was performed in 30 µl in the same conditions as the fluorescence assay using 4 µM [ 14 C]lipid II (0.06 µCi / nmol) instead of the fluorescent substrate and by omitting the muramidase. The reaction was incubated 15 min at 30°C, stopped by heating at 80°C for 10min and the products were separated by thin-layer chromatography (TLC) on silica gel plates in 2-propanol-ammonium hydroxide (25%)-H2O (6:3:1; V/V/V). The TLC plates were exposed to a storage phosphor screen (GE Healthcare) for 16 h, and images were revealed using a Typhoon Trio imager and Image Quant TL software (GE Healthcare).

The GTase inhibition assay was carried out by measuring the residual activity (RA) of E. coli PBP1b in the presence of 400 and 800 μM compounds. All the assays were repeated at least three times. The IC50 values were determined for the active compounds using variable concentrations of inhibitor (0-1000 μM) by fitting the data using GraphPad Prism 6.0 software.

Fluorescent anisotropy (FA) assay

Fixed concentrations of NBD-lipid II (0.33 µM) and PBP1b (~1 µM) in the 25 mM Tris-HCl pH 7.5, NaCl 0.1 M, CHAPS 0.14 % w/v, were mixed with serial dilutions of the compounds in 384-well plates. All the experiments were performed in triplicates and blank samples without proteins were also prepared for background fluorescence estimation. The mixtures (30 µL) were allowed to reach equilibrium for 20 min at room temperature with gentle shaking and the FA signals were recorded at 21°C using an Infinite® F Plex (Tecan, Männedorf, Switzerland) microplate reader equipped with polarization filters with excitation and emission wavelengths at 485 nm and 535 nm respectively. For direct binding evaluation of the compounds with the probe, the experiments were performed in the same condition in the absence of the protein. FA values in millianisotropy units (mA) were plotted as a function of compound concentrations in µM as previously described [START_REF] Boes | Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ[END_REF].

  . The samples (50 µl) contained 10 µM dansyl-lipid II, 50 mM HEPES-NaOH pH 7.5, 200 mM NaCl, 10 mM CaCl2, 0.085% of decyl-PEG, 20 % of dimethylsulfoxide (DMSO), 1 unit of N-acetylmuramidase of Streptomyces globisporus (Sigma) and variable concentrations of compounds. The reactions were initiated by the addition of 100 nM PBP1b and monitored by following the fluorescence decrease over 15-20 min at 30°C using an Infinite 200 PRO Microplate reader (Tecan, Männedorf, Switzerland) with excitation wavelength at 340 nm and emission at 520 nm.

Figure legend Figure 1 .

 1 Figure legend

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

20

 20

Figure 4 .

 4 Figure 4.

Table 1 .

 1 GTase inhibition activities of squalamine and aminosterol analogues. RA = the residual GTase activities values of PBP1b in % of inhibition compared to non-treated control are shown for two compounds concentrations (400 and 800 µM). Both RA and IC50 data represent mean values of three independent experiments ± s.d.nd, not determined.

  C NMR (75 MHz, MeOD):  = 71.65, 59.88, 58.54, 56.29, 56.04, 54.75, 48.18, 47.29, 42.71, 42.64, 39.94, 39.50, 39.04, 36.16, 35.78, 35.65, 31.56, 31.03, 30.40, 29.67, 27.99, 25.96, 24.35, 23.81, 22.79, 22.54, 21.05, 18.65, 16.33,

	14.09, 12.15. C31H58N2O ; MS (ESI) m/z = 475.4 [M+H] +

(13S,17S)-17-((4-aminobutyl)amino)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-

  1H). -13 C NMR (75 MHz, MeOD):  = 140.88, 125.41, 55.81, 55.47, 55.07, 52.16, 47.41, 47.13, 47.02, 42.85, 40.71, 40.61, 40.45, 39.50, 39.40, 38.40, 37.52, 37.25, 36.22, 35.70, 32.97, 32.87, 30.36, 30.26, 29.05, 28.87, 28.19, 24.78, 24.24, 24.14, 24.06, 22.66, 21.32, 18.76, 18.65, 12.35. C36H68N4 ; MS (ESI) m/z = 557.54 [M+H] +

	-1.25 (m, 4H), 1.27-1.35 (m, 8H), 1.36-
	1.55 (m, 8H), 1.57-2.03 (m, 13H), 2.31-2.37 (m, 1H), 2.59-2.87 (m, 7H), 2.94-3.01 (m, 2H),
	5.36-5.38 (m,

  . C22H34N2O, M = 342.53 g.mol -1 , Calc. C 77.15, H 10.1, N 8.18, Exp.C 77.12, H 11.3, N 8.21.

Acknowledgements

This work was supported by the "Fonds de la Recherche Scientifique" CDR J.0030.18. MT is a research associate of the FRS_FNRS (Brussels, Belgium), AB is supported by FRIA 1.E.038.17 (Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture) fellowship FRS_FNRS.