Placental BDNF/TrkB Signaling System is Modulated by Fetal Growth Disturbances in Rat and Human

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Human tissue collection
  • 2.2. Quantitative RT-PCR
  • 2.3. Animal model and tissue collection
  • 2.4. Semi-quantitative RT-PCR
  • 2.5. In situ hybridization
  • 2.6. Placental BDNF immunoassay
  • 2.7. Western blot analysis of placental TrkB-FL and TrkB-T1 receptors
  • 2.8. Statistical analysis
• 3. Results
  • 3.1. Effects of IUGR, fetal overgrowth and maternal type 1 diabetes on the gene expression level of BDNF and its receptors in the human placenta
  • 3.2. Effects of a 50% maternal food restriction on fetal and placental weights at term in rat
  • 3.3. Effects of maternal FR50 on mRNA and protein expression levels of BDNF and TrkB receptors in the rat placenta at term
• 4. Discussion
• Acknowledgements
• References
• Copyright

Abstract 

The brain-derived neurotrophic factor (BDNF) has been shown to exert an important role during implantation, placental development, and fetal growth control in mice. Its expression is closely related to the nutritional status in several tissues such as in the nervous system. In a previous study, we demonstrated that maternal undernutrition (MU), during the perinatal life, modified both the BDNF and its functional receptor, the tyrosine kinase receptor B (TrkB) gene expression in the brain of growth-restricted rat offspring during sensitive developmental windows, suggesting that these early modifications may have long-lasting consequences. In the present study, we measured BDNF/TrkB mRNA and protein levels in rat placentas from mothers submitted to a 50% food restriction during gestation, and in human placentas from pregnancies with fetal growth restriction or fetal macrosomia. In the rat, two subtypes of placental TrkB receptors have been identified: the TrkB-FL and TrkB-T1 receptors. We found that MU induced intrauterine growth restriction (IUGR) of fetuses at term and decreased the placental BDNF mRNA and protein levels. Placentae from undernourished mothers exhibited an increased mRNA expression of TrkB-FL whereas both TrkB-FL and TrkB-T1 receptors proteins levels were not modified. In human IUGR placentas, both BDNF and TrkB receptor mRNA expressions were up-regulated. Finally, although neither BDNF nor TrkB mRNA levels were altered by fetal macrosomia alone, BDNF mRNA levels were decreased when macrosomia was associated with maternal type 1 diabetes. These results show that the placental BDNF/TrkB system is modulated in rats and humans during pregnancies with fetal growth perturbations and is affected by the maternal energetic status. These data suggest that this system may exert an important role for the feto-placental unit development and that it may also be implicated in the etiology of pathologies related to placental and fetal growth disturbances.

Keywords: BDNF, TrkB receptor, Placenta, Fetal growth, Rat, Human

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1. Introduction 

Genetic background and environmental factors, such as nutritional and hormonal conditions are involved in the velocity of fetal growth [1]. Changes that influence the supply of nutrients to the fetus may alter its growth trajectory, as exemplified by fetal overgrowth in diabetes due, in part, to excess glucose whereas, food-restricted pregnancies result in intrauterine growth restriction (IUGR) [1], [2]. Accumulating experimental and clinical data indicate that disturbances of fetal growth trajectory resulting in the modulation of the birth weight is associated with increased rates of cardiovascular disease, hypertension, insulin resistance and type 2 diabetes later in life, a phenomenon termed fetal programming [3]. The major determinant of fetal growth is the placental supply of nutrients, a process that depends on the size, morphology, blood flow, and transporters abundance in the placenta [4]. As an example, lower placental weights are often associated with IUGR, maternal hypertension, or pre-eclampsia [5].

The placental development is temporally and spatially regulated by numerous molecules such as growth factors and cytokines, synthesized by different cell types at the materno-fetal interface and acting in autocrine, paracrine and/or juxtacrine manners [6], [7]. Recent evidence indicates that neurotrophins may also be implicated in the development of the placenta. In mice, it has been shown recently that the brain-derived neurotrophic factor (BDNF) exerts autocrine/paracrine roles in the placenta and potentiates both placental development and fetal growth from mid gestation to late gestational stages suggesting that this neurotrophin may be a crucial factor for the development of the feto-placental unit [8]. Biological effects of BDNF are mediated mainly through TrkB receptors. In addition to the full-length catalytic receptor (TrkB-FL), two truncated isoforms (TrkB-T1 and TrkB-T2) that are produced by alternative splicing of TrkB mRNA have been described in mammals [9]. Although lacking the intracellular tyrosine kinase activity, the TrkB-T1 and TrkB-T2 forms are also biologically active since they trigger transduction signals in several cells lines and under specific conditions [9], [10]. However, their physiological functions remain unclear. It has been suggested that these truncated receptors may have at least two main functions, i.e., acting as ligand trapping molecules to regulate the local availability of neurotrophins or functioning as dominant negative receptors of neurotrophin responsiveness by heterodimerization [9], [10]. At the placental level, it was demonstrated that the BDNF/TrkB signaling system increases trophoblast cell growth and survival during post-implantation periods especially in the labyrinth zone [8], a region composed of labyrinth trophoblasts with underlying blood vessels that provide a large surface area for nutrient, waste, and gas exchanges [11].

BDNF was originally found to be widely expressed in the central nervous system (CNS) where it exhibits a very high concentration in the hypothalamus and hippocampus [9]. In the brain, this neurotrophin influences almost all aspects of the CNS development while, in adulthood, it is rather involved in neuronal function, survival and differentiation [9]. In the CNS, a close relationship between nutritional status and BDNF level has been described. As an example, both the functional loss of one copy of BDNF or a de novo mutation of the full-length tyrosine kinase receptor B (TrkB-FL), the functional BDNF receptor, have been associated with severe obesity and developmental delay in human [12]. However, during development, little is known about the regulation of the BDNF/TrkB signaling system by the nutritional status in the nervous system and also in the other non neuronal tissues that express this neurotrophin. Recently, we have reported that maternal undernutrition modifies BDNF levels in the CNS of growth-restricted rat fetuses indicating that BDNF is sensitive to a reduction of the availability of nutrients and restriction of fetal growth [13]. In line with the reported contribution of BDNF both in the placental development and in the control of fetal growth, the present study aimed at investigating the placental BDNF/TrkB signaling system activity in IUGR rat fetuses from undernourished mothers. We also examined this system in placentas from human pregnancies with fetal growth complications such as IUGR or macrosomia. To this aim, we quantified the gene expression level of both BDNF and TrkB receptor in human placental samples from pregnancies with either IUGR, or fetal macrosomia, or maternal type 1 diabetes, or fetal macrosomia associated with maternal type 1 diabetes.

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2. Materials and methods 

2.1. Human tissue collection 

Sample collection of placental villi was performed under a protocol approved by the local institutional ethic committee. Placentas from normal and pathological pregnancies were collected after caesarean delivery from various maternities (Lille and Paris, France). Informed consent was obtained from all individuals for the use of placental samples. For the human study related to IUGR, four cases presenting with vascular IUGR and eleven uneventful pregnancies were analyzed. The inclusion criteria used for vascular IUGR were a weight at birth <10th percentile for a given gestational age and the observation of a Notch in uterine artery Doppler during the first and second trimesters without maternal pre-eclampsia. For the human study related to fetal macrosomia, five cases of each group (control and type 1 diabetes with or without macrosomia) were analyzed. Macrosomia was determined on the basis of standard growth curves for the French population [14]. Macrosomia was defined by the birth weight ≥ 90 th percentile according to gestational age and sex [14].

2.2. Quantitative RT-PCR 

RNA extractions have been performed in two different laboratories. For the human fetal IUGR study, RNA extraction was performed from placental villi using TRIzol reagent (Invitrogen Life Technology, Cergy, France). All RNA samples were treated by RQ1 DNase (Qiagen, France) in order to eliminate genomic DNA contamination (1 unit/μl for 20 μg of total RNA, in a final volume of 20 μl, at 37 °C for 30 min). For the human fetal macrosomia study, RNA extraction was performed using kit midi RNA/DNA (Qiagen, France). The cDNA conversion was performed as previously described [15]. Relative expression levels of RNA per sample were quantified by SYBR Green I assay on Roche Light Cycler 480 sequence detection assay (Meylan, France). For each transcript, PCR was performed in duplicates with 10 μl reaction volumes of 1 μl of cDNA, 5.5 μl of mix, 3.5 μl H₂O and 1 μl of primers set. The sequences of the primers for human BDNF were 5′-AGTGCCGAACTACCCAGTCGTA-3′ and 5′-CTTATGAATCGCCAGCCAATTC-3′, and for TrkB receptors 5′-CATTTCTCGAATCTCCAACCTCAGA-3′ and 5′-CTGGGCCTTTCATGCCAAACTTGGA-3′. Cyclo B was used as an internal standard as previously described [15]. The primers used for all genes were designed to span an intron to avoid genomic DNA amplification and contamination. PCR was conducted using the following cycle parameters: 2 min at 50 °C, 10 min at 95 °C and 40 three steps cycles of 15 s at 95 °C, 20 s at 50 °C and 20 s at 72 °C. The assay was performed following the manufacturer’s recommendations except that the reaction volume was reduced to 10 μl. A pool of cDNA from control placental tissues prepared immediately after partum was used as a standard (in threefold serial dilutions) for quantitative correction. All cDNA samples were used in dilution of 1:5 to obtain results within the range of the standard. Each sample was evaluated in duplicate. Analysis of transcript level was carried out using first the determination of the threshold cycle Ct for each reaction corrected by the efficiency. Then the delta Ct was calculated by subtracting the mean Ct of the calibrator from each value of Ct for each gene. The amount of target relative to a calibrator was computed by 2^{−delta Ct}. A previous validation by determination of amplification efficiency for each target and calibrator genes and a confirmation that these efficiencies are comparable between genes have been performed in our laboratory.

2.3. Animal model and tissue collection 

The experiments were conducted in accordance with the European Communities Council Directive of 1986 (86/609/EEC). Animal use accreditation by the French Ministry of Agriculture (No. 04860) has been granted to our laboratory for experimentation with rats.

Adult Wistar rats were purchased from Charles Rivers Laboratories (L’Arbresle, France) and housed (five per cage) with a controlled light cycle (12-h light/dark cycle, light on at 07 am) and temperature (22 ± 2 °C) with free access to food (regular rat chow No. A04, containing 22% protein, 5% fat, 53% carbohydrates; UAR, Villemoisson-sur-Orge, France) and tap water. After 14 days of acclimation, females were mated with a male for 1 night. Day 0 of pregnancy or embryonic day 0 (E0) was defined as the day immediately following the night during which males were present if spermatozoa were found in the vaginal smears. Pregnant rats were then housed in individual cages and fed ad libitum.

Two groups of pregnant rats were studied. In the control group (C group; n = 6), dams were fed ad libitum during gestation (from E0 to E21). In the food-restricted group (FR50 group; n = 6), females received from E14 to E21, 50% of the food-intake of control mothers, which has been previously determined in a series of pilot studies (FR50 females received 12 g/day of food from E14 to E21). The placentas and fetuses were collected at E21 by caesarean section after decapitation of pregnant mothers between 9 and 10 am. The placentas and fetuses were weighed and the sex was determined in fetuses just before their decapitation. Each litter contained between 8 and 13 fetuses. Experiments were conducted only on tissues from male fetuses. For each measurement, only one placenta/litter was used to obviate a putative litter effect. For fetal and placental weights analysis, litters have been averaged for these weights and then these averages have been used for comparison between groups. Placentas were frozen in liquid N₂ and stored at −80 °C until BDNF assays or semi-quantitative RT-PCR experiments. For in situ hybridization, placentas from the control group (n = 4) were frozen on dry ice and stored at −80 °C until sectioning.

2.4. Semi-quantitative RT-PCR 

Frozen rat placentas were homogenized at 4 °C using a polytron (PowerGen 700; Fisher Scientific, Pittsburgh, PA, USA) using a 10 × 195 mm sawtooth generator probe for 1 min at 15,000 rpm. RNA was extracted and purified (n = 6 placentas/group) using the TRIzol reagent (Gibco BRL, Strasbourg, France). The quality of total RNA was assessed by determining the 260/280-absorbance ratio and by agarose gel electrophoresis. The semi-quantitative RT-PCR method used here has been described and validated previously [16].

Briefly, 3 μg of total RNA were reverse transcribed into cDNA using 3 μg of random hexamers and 200 U Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (RT) (Gibco BRL, Strasbourg, France). One thirtieth of the first strand synthesis reaction was amplified using 1 U Taq DNA polymerase (Qbiogen, Illkirch, France) and 2 μM of each forward and reverse primers. The cycling parameters were: 94 °C for 1 min 30 s, 60 °C for 1 min 30 s, and 72 °C for 2 min. Negative control RT-PCR reactions were performed by omitting cDNA from the reaction mixture. The sequences of the rat primers for cyclophilin B (cyclo B) were 5′-GGAGACGAACCTGTAGGACGAGTGA-3′ and 5′-CTTGCCACAGTCTACAATGATCACA-3′, and for BDNF were 5′-GCGGACTTGTACACTTCCCGGGTGA-3′ and 5′-TCTATCCTTATGAACCGCCAGCCAA-3′ for forward and reverse primers respectively. For the specific detection of two isoforms of the TrkB receptor (TrkB-T1 and TrkB-T2), a forward primer (5′-CATTTCTCGAATCTCCAACCTCACA-3′), directed against the extracellular segment that is common to these isoforms was used. Separate isoform specific reverse primers, directed against the intracellular domain, allowed for the specific detection of the two isoforms of the TrkB receptor (5′-CTACCCATCCAGGGGGATCTTATGA-3′ for TrkB-T1 and 5′-AGCAAAAT AAGCACACTTCTGCTTA-3′ for TrkB-T2). The sequences of the rat primers for TrkB-FL were 5′-GCTGTCATCATTGGGATGACCAAGA-3′ and 5′- GGCGGGTTACCCTCTGCCATCAGCA-3′. Cyclo B was used as an internal standard. The primers used for all genes were designed to span an intron to avoid genomic DNA amplification and contamination. Each experiment was performed in triplicates and gave similar results. After amplification, the samples were separated on a 2% agarose gel, visualized by ethidium bromide and quantified by the Multi-Analyst (Biorad Laboratories, Hercules, CA) software.

2.5. In situ hybridization 

Sections (12 μm-thick) of some control placentas (n = 4) were mounted on gelatine-coated slides, dried and kept at −80 °C. In situ hybridization was performed as previously described [17]. BDNF and TrkB cDNAs for the detection of the three isoforms of the TrkB receptor (kindly supplied by Dr. F. Rage, Institut de Génétique Moléculaire, France) were subcloned into pGEM-T easy. The BDNF probe, a 250-bp fragment, and the TrkB probe, a 550-bp fragment, were linearized and labelled using [³⁵S]-dUTP (1300 Ci/mmol, Amersham Biosciences, Germany) with the Sp6/T7 Transcription Kit (Roche Diagnostics, Germany). Controls included hybridization with sense probe; no specific hybridization signal was observed under these conditions for both sense probes. For each probe, the slides were exposed together on one X-ray film (Biomax-MR, Kodak, France). Autoradiograms were digitized during the same session.

2.6. Placental BDNF immunoassay 

BDNF protein was measured in whole E21 rat placentas with a conventional two-site enzyme-linked immunosorbent assay as previously described [13]. The BDNF E_max immunoassay (Promega, Charbonniere, France) was performed according to the manufacturer’s protocol. Each placenta was placed in 500 μl of 100 mM Tris–HCl, 400 mM NaCl, 4 mM ethylenediaminetetraacetic acid, 0.2 mM phenylmethanesulphonyl fluoride, 0.2 mM benzothonium chloride, 2 mM benzamidine, 1.25 μg/mL aprotinin, 0.05% sodium azide, 2% bovine serum albumin, 0.5% gelatine and 0.2% Triton X-100. Tissues were briefly sonicated and the homogenates centrifuged for 20 min at 10,000 × g at 4 °C. A 100-μl fraction of supernatant was used to determine the BDNF content. The assay sensitivity was 15 pg/mL and the cross reactivity with other related neurotrophic factors was <3%. The BDNF concentration was expressed as pg/g of wet weight of tissue. The intra- and inter-assay coefficients of variation were 4% and 5%, respectively.

2.7. Western blot analysis of placental TrkB-FL and TrkB-T1 receptors 

In rat placentas, only TrkB-FL and TrkB-T1 receptors (which mRNAs have been detected using RT-PCR) have been assayed using Western blot. The antibodies used for TrkB-FL (sc-12, lot J111) and TrkB-T1 (sc-119, lot I1004) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Western blot analysis was performed as previously described [18]. Briefly, 500 μl of cold lysis buffer (150 mM NaCl, 1.5 mM MgCl₂, 10% glycerol, 5 mM EDTA, 50 mM HEPES, pH 7.5 supplemented with 1 mM dithiothreitol, 1 mM leupeptin, 1 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride and 1 mM orthovanadate) were added and the samples sonicated for 4 × 5 s using an immersed probe. A post-nuclear supernatant was obtained after 10 min centrifugation at 380 × g. The supernatant membranes were solubilized by adding 1% Triton (X-100) and 0.2% SDS and gently rocked for 30 min at room temperature. Unsolubilized material was pelleted by centrifugation (15,000 × g for 15 min at 4 °C) and discarded. Protein concentrations were determined by spectrophotometry. Proteins (200 μg) were mixed 1:1 with sample buffer (10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.05% bromophenol blue) and boiled for 5 min. The samples were separated on 7.5% SDS-polyacrylamide gels and transferred electrophoretically onto a nitrocellulose membrane. For detection of antibody immunoreactivity, nitrocellulose membranes were incubated in Tris-buffered saline with 0.1% Tween 20 (TBST) supplemented with 5% skimmed milk powder and 2% bovine serum albumin for 30 min and then soaked overnight at 4 °C in TBST supplemented with 2% skimmed milk powder with the appropriate antibody: rabbit polyclonal anti-TrkB-T1 at a 1:500 dilution. With the rabbit polyclonal anti-TrkB-FL at 1:200 dilution, nitrocellulose membranes were incubated in the same buffer for 1 h at room temperature and then overnight at 4 °C. After primary antibody incubation, membranes were washed three times with TBST and incubated for 1 h at room temperature with an anti-rabbit IgG antibody (Amersham, Orsay, France) conjugated to horseradish peroxidase at a 1:1000 dilution, followed by the enhanced chemiluminescence method on the Lumi-Imager F1 system (Roche). Protein bands were quantified by image analysis with the Lumi Analyst system. The nitrocellulose membranes were also soaked with a rabbit polyclonal anti-actin (Sigma, St. Louis, MO, USA) at a 1:200 dilution to ensure equivalent amounts of loaded proteins.

2.8. Statistical analysis 

All data are presented as the mean ± SEM. Statistical analysis was performed using one way ANOVA and post-hoc comparison by Dunnett’s test. P < 0.05 was considered statistically significant. Analyses were performed using SigmaStat software (Systat Software, Port Richmond, CA, USA).

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3. Results 

3.1. Effects of IUGR, fetal overgrowth and maternal type 1 diabetes on the gene expression level of BDNF and its receptors in the human placenta 

The body weight of IUGR newborns was reduced by 13% (P < 0.001) and their gestational age were also reduced i.e. 33.6 vs 38.6 weeks in controls (P < 0.05; Table 1A). Fetal macrosomia increased both newborn (+29.3% in the C + M group and +11.2% in the D + M group compared to respective controls) and placental weights (+54.5% in the C + M group and +5.1% in the D + M group compared to respective controls; Table 1B). The gestational age of newborns with or without macrosomia from control or diabetic mothers reported in the Table 1B did not differ by more than 2.5 weeks. The expression of BDNF and TrkB receptors in human placentas was determined by quantitative RT-PCR. In IUGR pregnancies, both the BDNF and the TrkB receptors mRNA levels were drastically increased respectively 2-fold (P < 0.01) and 8-fold (P < 0.05) versus controls (Fig. 1A & B). The BDNF mRNA levels were significantly (P < 0.05) reduced in pregnancies with both maternal type 1 diabetes and fetal overgrowth in comparison to control pregnancies with or without fetal macrosomia and, pregnancies associated with maternal type 1 diabetes (Fig. 1C). Finally, fetal overgrowth alone, maternal type 1 diabetes, and the association of both maternal type 1 diabetes and fetal overgrowth did not significantly modify TrkB receptors mRNA levels at term (Fig. 1D).

Table 1. Human maternal and fetal parameters.

Values are means ± SEM (n = 4–11 mothers or babies/group in Table 1A).

Values are means ± SEM (n = 5 mothers or babies/group in Table 1B).

C: control, M: fetal macrosomia, D: maternal type 1 diabetes.

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• Fig. 1 

  Quantitative RT-PCR of BDNF (A, C) and TrkB (B, D) mRNA levels in human placentas. C: control (n = 11 in A and B; n = 5 in C and D); IUGR: pregnancies with fetal growth restriction (n = 4 in A and B); C + M: pregnancies with fetal overgrowth (n = 5); D: pregnancies with maternal type 1 diabetes (n = 5); D + M: pregnancies with maternal type 1 diabetes and fetal overgrowth (n = 5). *P < 0.05, **P < 0.01 IUGR group versus C group (in A and B). Maternal and newborn’s parameters are reported in Table 1.

3.2. Effects of a 50% maternal food restriction on fetal and placental weights at term in rat 

At E21, the maternal FR50 diet reduced maternal weight of 28% (P < 0.001; Table 2). The weight of FR50 fetuses was also decreased (reduction of 15%; P < 0.001) but no modification of the placental weight was noted. Finally, a significant reduction of the fetus/placenta ratio was observed in the FR50 group (P < 0.001; Table 2).

Table 2. Maternal and fetal parameters.

Values are means ± SEM [n = 6 litters/group].

3.3. Effects of maternal FR50 on mRNA and protein expression levels of BDNF and TrkB receptors in the rat placenta at term 

The localization BDNF and TrkB receptors mRNAs was determined by in situ hybridization. In control rats, BDNF mRNAs were detected in the placental basal zone while TrkB receptor mRNAs were detected in the trophospongium (a part of the basal zone) and in areas close to the labyrinth (Fig. 2). Placental BDNF mRNA and protein expression levels of were reduced by 46% and 16% respectively in FR50 rats (P < 0.05; Fig. 3). Using RT-PCR, we detected two isoforms of the TrkB receptor in rat placentas: the TrkB-FL and the TrkB-T1 receptors (data not shown). In FR50 placentas, the mRNA expression level of the TrkB-FL was significantly increased (P < 0.05; Fig. 4A) but without modification of its protein level (Fig. 4B). Finally, no modification of either mRNA or protein levels of the TrkB-T1 receptor was observed (Fig. 4C and D).

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• Fig. 2 

  Photomicrograph of a coronal section showing specific in situ hybridization signal (for anti-sense probes) for BDNF mRNA (A) and TrkB mRNA (B) in a placenta of E21 control rat. Labeling is visible in black and indicated by large black arrows. No signal was detected using sense probes (data not shown). Lab: labyrinth; BZ: basal zone; Dec: decidua; Fv: fetal vessels. Magnification bar = 3 mm.

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• Fig. 3 

  Semi-quantitative RT-PCR analysis of BDNF mRNA (A) and BDNF protein levels (B) in control (white bars) and FR50 (dark bars) E21 rat placentas (n = 6 animals per group). *P < 0.05 FR50 versus C group.

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• Fig. 4 

  A. Semi-quantitative RT-PCR analysis of mRNA level (A, C) and Western blot analysis of protein level (B, D) of the two TrkB isoforms (TrkB-FL and TrkB-T1) in E21 rat placentas. (n = 6 animals per group). *P < 0.05 FR50 versus C group.

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4. Discussion 

In the present study, we report that the placental BDNF/TrkB signaling system is modulated in pregnancies with fetal growth perturbations in both rat and human. IUGR decreases the expression of the placental BDNF/TrkB system in rat but increases it in human. Maternal type 1 diabetes associated or not with fetal macrosomia also decreases its expression in human pregnancies. Altogether, these results suggest that this system usually implicated in the activity of the nervous system may be an important contributor to the control of fetal growth at the placental level. In the rat, maternal undernutrition (MU), performed during late gestation, reduces both BDNF mRNA and protein levels at term, demonstrating that the endogenous placental production of this neurotrophin is altered in IUGR rat fetuses as a possible consequence of maternal undernutrition. The localization of placental BDNF gene expression in control rats was found to be close to the basal zone, the major site of placental hormone production [19]. This localization is in accordance with its recently reported distribution in the mouse placenta [8] showing that BDNF immunoreactivity is strongly detected in spongiotrophoblasts and decidual cells at the end of gestation. However, at an early stage of gestation in mice (day 10 of pregnancy), a transient strong BDNF expression was also observed in the labyrinth, the major site of feto-maternal exchange [8]. In the rat placenta, TrkB receptor gene expression was found to be located in the trophospongium and in a zone close to the labyrinth. In accordance, in mouse, TrkB-FL receptor immunoreactivity has been detected in labyrinth trophoblasts but also in decidual cells and a weaker staining was also found in spongiotrophoblasts [8]. Such discrepancies between rat and mouse may reflect differences in the techniques used but also putative slight specie-specific site of expression. For the first time, we reported that the rat placenta expressed two types of TrkB receptor: the TrkB-FL and the truncated isoform TrkB-T1. MU did not affect the protein levels of these receptors at term, but increased TrkB-FL mRNA level in IUGR rat fetuses. Studies performed in vitro and in pregnant mice [8] have reported that treatment of cultured trophoblast cells with the TrkB ectodomain, or a Trk receptor inhibitor (i.e. K252a), suppresses cell proliferation and increases apoptosis. Moreover, in vivo, K252a administration to pregnant mice suppressed placental development and was accompanied by an increase in trophoblast cells apoptosis and a decrease in labyrinth zone volume at midgestation. Taken together these data may suggest that, in growth-restricted fetuses from food-restricted mothers, the reduction of placental BDNF level may increase the placental apoptosis. In accordance with this hypothesis, it has been recently shown that rat placentas from 50% food-restricted mothers during 10 days of pregnancy (from E10 to E20 of gestation) presented significantly higher levels of apoptotic indices in both basal and labyrinth zones [20] suggesting that the functions of these placental structures may be altered. Thus, as the labyrinth represents the main maternal/fetal interface exchange zone, we can postulate that nutrients transfer may be disturbed in FR50 fetuses. Moreover, an alteration of this zone in FR50 rats may also induce a defective placental barrier that could explain, in part, our previous finding of increased transplacental transfer of maternal glucocorticoids in this model [17].

Finally, in addition to an endogenous role of TrkB receptors in the rat placenta for the control of cells proliferation/survival, we postulate that these receptors may also be implicated in the transplacental transfer of neurotrophins i.e. BDNF but also neurotrophin-4/5 (NT-4/5), the second natural ligand of TrkB receptors. Indeed, in the mouse, it has been shown that maternal BDNF is able to reach the fetal brain through utero-placental barrier and may therefore contribute to the development of fetal CNS [21]. However, the mechanism of such transport remains unknown. In agreement with our hypothesis, Trk receptors have been implicated in the transport of neurotrophins in the CNS [22]. Thus, we can postulate that in trophoblast cells BDNF may be transported to fetal blood and/or amniotic fluid through TrkB interaction and transport through transcytosis after both ligand endocytosis (at the level of the maternal compartment), transcellular transport and then ligand exocytosis (to the fetal compartment). However, the intensity of such putative transplacental passage of BDNF and NT-4/5 from blood may be physiologically modest at term as very low levels of these neurotrophins have been reported in both mouse and human maternal plasmas during gestation [8], [23]. Nevertheless, the placenta may likely be the major source of BDNF production to the amniotic fluid during pregnancy [23] and this neurotrophin may thus exert putative physiological actions on the fetus. Finally, it could also be speculated that placental BDNF may be implicated in angiogenesis of the feto-placental unit. Indeed, after its original discovery such as a neurotrophic factor, BDNF has also been shown to affect vascular cell processes. Molecules that have such dual properties have been referred to as angioneurins [24]. For example, genetic studies in mice revealed that BDNF is required for the maintenance of cardiac vessel-wall stability during development [25]. Moreover, in the adult, both BDNF and NT4 stimulate angiogenesis in the heart, skeletal muscle and skin by binding to TrkB receptors on endothelial cells [26].

In human placentas, we quantified the gene expression level of both BDNF and TrkB receptor in placental samples from pregnancies with fetal growth disturbances to look at a putative modulation of the BDNF/TrkB signaling system. The maternal gestation age for the placental samples from IUGR condition that we used here were slightly different but it has been previously reported that the gene expression profile of both BDNF and TrkB was not affected from midgestation to term in the basal plate of human placentas [27]. These data suggest that the gestational age may not drastically affect the BDNF/TrkB system expression during late gestation but we cannot exclude a specific modulation of this system in villous tissue. In contrast to IUGR rats from FR50 mothers, IUGR in human increases significantly both BDNF and TrkB receptor gene expression suggesting that this system may be a regulatory mechanism required to counterbalance the restriction of fetal growth if, as in rodent, this system exerts a trophic action on the feto-placental unit. In accordance with this hypothesis, we did not detect any modification of this system in condition of fetal macrosomia suggesting that the BDNF/TrkB system is rather implicated in the maintenance of growth and development under deleterious conditions. In humans, we also show that the maternal metabolic status is implicated in these regulations as we noticed a reduction of the expression of this system in pregnancies with both maternal type 1 diabetes and fetal macrosomia. The maternal glucose metabolism may represent an important regulator of placental BDNF gene expression as a recent study has reported, using a microarray study (representing 22,000 genes investigated), that the placental BDNF expression is up-regulated in gestational diabetes mellitus, among only 66 genes differentially expressed [28]. In humans, the placental BDNF/TrkB signaling system seems to be rather implicated in the maintenance of placental and/or fetal growth under deleterious conditions and, as demonstrated in rat, the maternal energetic status seems to be a critical regulatory factor of this system.

Herein, we have demonstrated a modulation of the placental BDNF/TrkB signaling system during pregnancies with maternal undernutrition, type 1 diabetes and disturbances of fetal growth. These findings reinforce previous observations and suggest important roles of this system for both placental development and the control of adequate fetal growth. Further investigations of the physiological actions of neurotrophins at the placental level may lead to new understanding on the management of abnormal pregnancy and mechanisms of fetal programming. We also postulate that the placental production of neurotrophins, such as BDNF, may be implicated in the establishment and maturation of the fetal nervous system. Thus, the placental production level of BDNF may be potentially implicated, at an early stage, in some mechanisms of defective programming of the CNS in IUGR offspring as previously described [29]. Such putative functions of placental neurotrophins have also been suggested to be implicated in the deleterious CNS long-lasting consequences of maternal infection [30], [31].

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Acknowledgements 

This work was carried out with the financial support of the “ANR- Agence Nationale de la Recherche – The French National Research Agency” under the “Programme National de Recherche en Alimentation et nutrition humaine”, project “ANR-06-PNRA-022” and the Fondation Cœur et Artères (FCA N° 05-T4).

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