ORGAN TOXICITY AND MECHANISMS
Developmental exposure to nonylphenol induced rat axonal injury in vivo and in vitro
Siyao Li1 · Mingdan You1 · Wenjie Chai1 · Yuanyuan Xu2 · Yi Wang1
Received: 14 May 2019 / Accepted: 14 August 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Increasing evidence indicates that developmental exposure to nonylphenol (NP) causes damage to the central nervous system (CNS). As the most unique and primary component of neuron, axon is an essential structure for the CNS function. Here, we investigated whether developmental exposure to NP affected rat axonal development in vivo and in vitro. Our results showed that developmental exposure to NP 10, 50, and 100 mg/(kg day) caused an obvious decrease in axonal length and density in the hippocampus. Developmental exposure to NP also altered the expression of CRMP-2 and p-CRMP-2, and activated Wnt-Dvl-GSK-3β cascade in the hippocampus, the crucial signaling that regulates axonal development. Even months after the exposure, impairment of axonal growth and alteration of this cascade were not fully restored. In the primary cultured neurons, 30, 50, and 70 μM NP treatment decreased axonal length and impaired axonal function. Similar to in vivo results, it also activated Wnt-Dvl-GSK-3β cascade in cultured neurons. SB-216763, a specific GSK-3β inhibitor, recovered the shortening of axon and the impairment of axonal function induced by NP. Taken together, our results support the idea that exposure to NP induces axonal injury in the developing neurons. Furthermore, the activation of Wnt-Dvl-GSK-3β cascade contributes to the axonal injury induced by NP.
Keywords Nonylphenol · POPs · Axon · Wnt-Dvl-GSK-3β cascade
Abbreviations
BBB Blood–brain barrier
CNS Central nervous system
CRMP-2 Collapsin response mediator protein-2 DMSO Dimethyl sulfoxide
Dvl Dishevelled
GD Gestational day
GSK-3β Glycogen synthase kinase-3β NP Nonylphenol
NPnEO Nonylphenol ethoxylates PB Placental barrier
PND Postnatal day
POPs Persistent organic pollutants p-CRMP-2 Phosphorylated CRMP-2
p-GSK-3β Phosphorylated GSK-3β
SB SB-216763
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00204-019-02536-0) contains
Imagesupplementary material, which is available to authorized users.
* Yuanyuan Xu [email protected]
* Yi Wang
[email protected]
1 Department of Occupational and Environmental Health, School of Public Health, China Medical University, No 77 Puhe Road, Shenyang North New Area, 110122 Shenyang, Liaoning, People’s Republic of China
2 Program of Environmental Toxicology, School of Public Health, China Medical University, No 77 Puhe Road, Shenyang North New Area, 110122 Shenyang, Liaoning, People’s Republic of China
Introduction
Nonylphenol (NP), the final catabolite of nonylphenol eth- oxylates (NPnEO), is a typical persistent organic pollutant (POP). Large volumes of NPnEO are used in a variety of agricultural, industrial, beauty, and especially household products (Brunelli 2018; Zhang et al. 2015). As a conse- quence, NP has been found in various environmental media, and people are largely exposed to this priority hazardous substance. The main routes of human exposure to NP are through food and drinking water (Arukwe et al. 2000; Car- eghini et al. 2015; Lee et al. 2015; Lu et al. 2007). Previous
reports have documented the accumulation of NP in diverse tissue of humans (Calafat et al. 2005; Inoue et al. 2000; Lopez-Espinosa et al. 2009). Remarkably, a study of preg- nant and lactating women in Taiwan demonstrated that NP was detected in maternal blood, fetal cord blood, placenta, and breast milk (Huang et al. 2014). The intact form of NP at environmentally relevant concentrations could transfer across the human placenta (Balakrishnan et al. 2011). In line with this, a study of Indian women showed the highly positive correlation between the concentrations of NP in maternal blood and in amniotic fluid (Shekhar et al. 2017). Furthermore, this study also indicated the highest mater- nal–fetal transfer of NP among various phenolic POPs.
It is well known that NP exposure has adverse effects on animals and humans, including general toxicity and repro- ductive toxicity (Jie et al. 2013a; Kazemi et al. 2016a, b). Notably, increasing evidence indicates that NP exposure potentially damages the structures and functions of central nervous system (CNS). It has been shown that NP exposure altered motor co-ordination, grip strength, and cognitive performance of rats, and caused neurochemical and histo- pathological perturbations in brain tissues (Tabassum et al. 2017). Kazemi et al. reported a positively linear correlation between NP concentration in specific brain areas and behav- ioral impairments of rats (Kazemi et al. 2018). Another study showed that oral administration of NP impaired spatial learning and memory, as well as fear-motivated learning and memory in rats (Kawaguchi et al. 2015). In addition, both in vitro and in vivo studies have reported that NP induced apoptosis in brain cells and neural stem cells (Kazemi et al. 2018; Kudo et al. 2004; Litwa et al. 2014, 2016). NP has also been reported to disturb neurogenesis and inhibit neuronal growth and differentiation (Jie et al. 2016; Kudo et al. 2004). There is a particular concern about the impact of maternal exposure to NP during developmental period on neurodevel- opment of offspring (Sise and Uguz 2017; Tsai et al. 2013). The main source of NP exposure for fetuses is transplacental absorption, while babies are also exposed to high levels of NP due to breastfeeding. Furthermore, a study in Germany indicated that babies’ daily intake of NP was also consider- able through some commercial baby food (Raecker et al. 2011). The brain is extremely sensitive to environmental insults during the developmental period (Rice and Barone 2000). It has been found that developmental exposure to some chemicals, such as metals, inorganic compounds, organic solvents, and certain POPs, causes impairments of learning and memory (Gillette et al. 2017; Grandjean and Landrigan 2006; Sobolewski et al. 2014). Notably, many lines of evidence have shown that developmental exposure to NP induces neurodevelopmental impairments, leads to behavioral alteration, and negatively affects learning and memory ability (Couderc et al. 2014; Jie et al. 2013b, 2016,
2017).
The most prominent hallmark of neuron is its single and slender axon (Son et al. 2012). As the highly specialized structure of neuron, it is believed to be the central com- ponent for connection of neural networks and intercellu- lar communication (Son et al. 2012). Axonal elongation is deeply implicated in the formation of neural network in the brain, which is necessary for cognitive function (Ken- nedy 2013). In addition, the polarization of axon, a widely accepted cellular basis for learning and memory, is required in information integrating and transmitting in the brain (Son et al. 2012). It is also known that axon is crucial for the generation and propagation of action potentials (Son et al. 2012). Furthermore, axon is considered as reliable trans- mission cable that essentially underpins nervous system function (Debanne et al. 2011; Prokop 2013). In the neural network, axon is dedicated to signal transfer over the signifi- cant distances, which also enables it to play a vital role in learning and memory and establish the information highway (de Anda and Tsai 2011).
Wnt-Dvl-GSK-3β cascade has been shown to mediate
axonal remodeling and guidance (Salinas 2005). The Wnt family of secreted proteins comprises signaling molecules that regulate cell polarity and migration, axonal morphol- ogy, synaptic differentiation, and so forth (Ciani et al. 2004; Peifer and McEwen 2002). Specifically, Wnt-7a is known to induce axonal spreading and guide synapse formation in neurons (Salinas 1999). Its downstream effector Dishevelled (DVL) stabilizes microtubules through the inhibition of gly- cogen synthase kinase-3β (GSK-3β), and thereby involves in axonal remodeling (Ciani et al. 2004). GSK-3β is a mul- tifunctional serine (Ser)/threonine (Thr) kinase that controls axon morphogenesis by mediating microtubule assembly, a necessary process of axonal extension (Zhou and Snider 2005). GSK-3β has a high level of basal kinase activity and its upstream signaling pathway usually functions by inhibit- ing the activity of GSK-3β. That is to say, the inactivation of upstream signaling often increases GSK-3β activity (Hur and Zhou 2010). The phosphorylation of GSK-3β at a single serine residue (Ser 9 at the N terminus of GSK-3β) is the principal mechanism for the inhibition of its activity (Stoica et al. 2016). Collapsin response mediator protein-2 (CRMP- 2) is crucial for axon formation, because it promotes micro- tubule assembly for neurite elongation; phosphorylation of CRMP-2 at Thr-514 by GSK-3β results in its inactivation (Yoshimura et al. 2005). The phosphorylated CRMP-2 lose the ability to bind to tubulin dimers, which impairs the estab- lishment and maintenance of neuronal polarity (Yoshimura et al. 2005).
The CNS is quite sensitive to some chemicals during
developmental period. Emerging evidence suggests that developmental exposure to NP causes the impairment of the CNS. Although axon is a basic and essential structure for the CNS function, the effect of developmental exposure
to NP on axon has not been investigated. Thus, the goal of this study was to investigate whether NP exposure during developmental period induced axonal injury. In addition, we also studied its effects on the development of axon in cul- tured neurons. Wnt-Dvl-GSK-3β cascade plays a key role in axonal growth. Hence, we explored whether this cascade was involved in the effects of developmental exposure to NP on axon both in vivo and in vitro in the present study.
Materials and methods
Reagents
NP (≥ 99.0%) was from Dr. Ehrenstorfer (Augsburg, Ger- many). Modified AIN-93G diet (a low phytoestrogen diet with 7% corn oil substituted for 7% soybean oil) was from HFK Bioscience Co., Ltd (Beijing, China). Corn oil from Sigma-Aldrich (St. Louis, MO, USA) was used as NP delivery vehicle. Neurobasal-A Medium, B-27 supplement, Penicillin–Streptomycin, Trypsin–EDTA, Dulbecco’s modi- fied Eagle’s medium (DMEM), and Hank’s Balanced Salt Solution (HBSS) were from GIBCO/Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was from Hyclone (Legan, UT, USA). β-D-arabinofuranoside, Poly-L-lysine, Glutamine, SB-216763 (SB), and Dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA). Pierce BCA protein assay reagent was from Thermo Scien- tific (Rockford, IL, USA). FM1-43, a styryl dye, was from Invitrogen (Eugene, OR, USA). Bielschowsky silver kit was from Yuan Mu Biotechnology (Shanghai, China). We used the following antibodies: anti-GSK-3β (Cell Signaling Tech- nology), anti-phospho-GSK-3β (Ser-9) (Cell Signaling Tech- nology), anti-CRMP-2 (Abcam), anti-phospho-CRMP-2 (Thr-514) (Abcam), anti-Dvl-1 (Santa Cruz Biotechnology), anti-Wnt (Proteintech), anti-GAPDH (Santa Cruz Biotech- nology), and anti-neurofilament (2H3) (Developmental Studies Hybridoma Bank).
Animals
Female and male Wistar rats, 8 weeks of age and weigh- ing 240–260 g, were obtained from the Center of Experi- mental Animals at China Medical University (Shenyang, China) with a National Animal Use License number of SYXK-LN-2013-0007. The Animal Use and Care Com- mittee at China Medical University approved all the ani- mal experimental procedures, which complies with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were housed (24 ± 1 °C, 50 ± 5% relative humidity, and 12 h light/dark cycle) and provided with modified AIN-93G diet and deionized water. Rats were housed for 1 week before entering the experiment.
The female rats were mated with male rats in a 1:2 pro- portion. The day of the vaginal plug detected was consid- ered gestational day 0 (GD0). Subsequently, the pregnant rats were randomly divided into control and NP treatment groups. 10–12 dams were in each group. Three NP treat- ment groups were given NP (dissolved in corn oil) daily by gavage at 10, 50, or 100 mg/(kg day), respectively, from GD0 till postnatal day 21 (PND21). The control group received the corn oil vehicle by gavage. All dams reared the pups until weaning on PND21. No observed adverse effect levels of NP were, respectively, reported from 10 to 50 mg/(kg day) in rats (Cunny et al. 1997; Jie et al. 2010, 2013a). In addition, some studies showed that 100 mg/ (kg day) NP treatment led to obvious impairments of nerv- ous system, without causing general toxicity (Jie et al. 2013b; Mao et al. 2008). Thus, the animals were treated with NP at 10, 50, or 100 mg/(kg day) in the present study, which did not cause obvious alteration in the general con- ditions, such as body weight.
Pups were randomly taken from different litters in each
group, and beheaded for hippocampal tissues, or perfused through the left ventricle using standard methods for pathological section on PND21 and PND80. Hippocam- pal tissues were isolated from brains and removed quickly into liquid nitrogen for protein extraction. Brains of per- fused pups were fixed overnight in the paraformaldehyde, embedded in paraffin, and sectioned into coronal sections. The sections were used for Bielschowsky silver staining and immunofluorescence.
Primary culture of neurons
Primary cortical neurons were prepared from newborn Wistar rats (< 24 h). Briefly, the whole cerebral cortex was rapidly isolated, and cells were dissociated in 0.125% trypsin at 37 °C for 20 min. The cell suspension was centrifuged at 1300 r/min for 5 min and re-suspended in DMEM medium containing 10% FBS and 1% peni- cillin–streptomycin. Then, cells were seeded onto pre- coated poly-L-lysine multi-well plastic culture palates at 37 °C in a humidified 5% CO2 atmosphere. After 24 h, the DMEM medium was replaced by Neurobasal medium supplemented with B-27, penicillin–streptomycin, and glu- tamine. After 48 h, cytosine β-D-arabinofuranoside was added into the medium to inhibit glial cell proliferation. From then on, half of the culture medium was replaced by fresh Neurobasal medium every other day. On day in vitro 7, neurons were treated with a final concentration (0, 30, 50, and 70 μM) of NP for 24 h. To further investigate Wnt- Dvl-GSK-3β cascade, GSK-3β inhibitor SB was used. SB was added into the cultures 30 min prior to NP exposure.
Western blot analysis
Hippocampal tissues or cultured neurons were homogenized and gently lysed in protein lysis buffer for 1 h, supplemented with phosphorylase inhibitors and protease inhibitors, and then centrifuged at 12,000 rpm for 10 min at 4 °C to obtain the supernatants. Then, supernatants were collected, and protein concentrations were determined using Pierce BCA protein assay. Protein extracts were analyzed by western blot according to the standard protocols. In brief, samples with equal amounts (30 μg per lane) of protein were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the pro- teins were transferred onto a PVDF membrane (Millipore, USA). After blocking in 5% bovine serum albumin (BSA) for 2 h, the membranes were incubated with specific primary antibodies overnight at 4 °C. Dilutions for primary antibod- ies were as follows: anti-GSK-3β (1:1000, Cell Signaling Technology), anti-phospho-GSK-3β (Ser-9) (1:1000, Cell Signaling Technology), anti-CRMP-2 (1:2000, Abcam), anti-phospho-CRMP-2 (Thr-514) (1:1000, Abcam), anti- Dvl-1 (1:1000, Santa Cruz Biotechnology), and anti-Wnt (1:1000, Proteintech). Membranes were washed three times with Tween 20/Tris-buffered saline (TBST) and incubated with a 1:5000 dilution of secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Membranes were washed three times with TBST again and detected by chemiluminescence system (Thermo Scientific). For each blot, the quantitative analysis was carried out by NIH Image J program and normalized to GAPDH in each sample.
Immunofluorescence analysis
Paraffin sections or neurons (adhered to glass coverslips) were subjected to immunofluorescence staining according to standard protocols. In brief, brain sections were washed three times in phosphate-buffered saline (PBS) after depar- affinization and rehydration. Neurons were cultured on poly- L-lysine-coated glass coverslips for immunofluorescence. At the end of treatment, neurons were fixed with 4% para- formaldehyde and permeabilized with Triton® X-100. Par- affin sections or neurons were blocked with 5% goat serum albumin for 1 h, and then incubated at 4 °C overnight in specific primary antibodies. Dilutions for primary antibod- ies were as follows: anti-neurofilament-specific antibody, 2H3 (1:50, Developmental Studies Hybridoma Bank) and anti-CRMP-2 (1:500, Abcam). Then, the sections or cells were washed in PBS and incubated in fluorescent second- ary antibody (Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488; or Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594) for 1 h at room temperature in the dark. After the
above operation, the sections were washed in PBS again. Photographs were obtained from a fluorescence microscope (DMIRB Microscope, Leica) or a fluorescence confocal microscope (A1R, Nikon).
Bielschowsky silver staining in hippocampus
Bielschowsky silver staining was conducted according to the manufacturer’s instructions. In brief, after deparaffini- zation and rehydration, the brain sections were incubated in silver nitrate solution at 37 °C for 30 min in the dark. Reducing agent was added to the silver nitrate solution until the sections turned yellow. Silver–ammonia solution was added to the sections for 30 s. Subsequently, reducing agent was added to the sections again. The slides were rinsed in gold chloride solution and fixed in sodium thiosulfate solu- tion. The images were obtained from a microscope (DMIRB Microscope, Leica) at a magnification of 200× to analyze the nerve fibers (black).
Analysis of axonal outgrowth in primary cultured neurons
As above description, neurons were immuno-stained with an antibody against 2H3, a well-known marker of axon. The analysis was performed to determine the length of neurites in vitro as previously described (Chou et al. 2001; Desai et al. 2009). The lengths of neurites were analyzed with NeuronJ plugin of ImageJ software (NIH, Bethesda, MD). For assessing the average length of 2H3 positive neurites per cell, the total length of neurites was divided by neuron numbers in the identical area obtained at a magnification of 200×. The images obtained at a magnification of 400× were used to calculate the length of the longest axon with ImageJ.
FM1‑43 loading and unloading in primary cultured neurons
The axonal function of cultured neurons can be visualized by the FM1-43 dye loading and unloading (Wegner et al. 2008; Zhao et al. 2013). Therefore, the FM1-43 staining experiment was performed, according to the manufacturer’s instructions. Briefly, neurons were stained with 10 µM FM1- 43 dye in depolarizing extracellular solution (45 mM KCl, 54 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM HEPES;
pH 7.3) for 3 min (loading) and washed with calcium-free Hank’s Balanced Salt Solution (HBSS) solution three times (5 min each). After excessive dye was washed off, FM fluo- rescent images were obtained. Subsequently, the same live neurons were subjected to 90 mM K+ solution for 5 min (unloading). The high level of K+ in this solution resulted in a release of the loaded dye, which can be used to evaluate the release function of the axons. FM fluorescent imageswere taken from 1 to 5 min after the addition of high K+ solution. Fluorescent images were captured with the inverted fluorescent microscope at a magnification of 200×(DMIRB Microscope, Leica).
Statistics
All experiments were performed in at least triplicate, and data were shown as mean ± SEM. Statistical analyses were performed using SPSS 16.0 software. Statistical significance was assessed by one-way analysis of variance followed by Student–Newman–Keuls test. P < 0.05 was considered sta- tistically significant.
Results
Developmental exposure to NP induced axonal injury in hippocampus
To explore the effects of developmental exposure to NP on nerve axons, we performed immunofluorescence to detect 2H3 in offspring hippocampus. 2H3 is a key member of neurofilament protein. Neurofilaments support the morphol- ogy of the axonal cytoplasm as cytoskeletal components and serve important roles in the regulation of axonal transport (Nixon and Sihag 1991). Compared with the control group, the level of 2H3 was obviously reduced in pups develop- mentally exposed to NP. Moreover, as shown in Fig. 1a, gestational and lactational exposure to NP led to an obvious decrease in axonal length and density in both CA1 and CA3 regions of pups on PND21 and PND80.
To further confirm the axonal injury induced by devel- opmental exposure to NP, we conducted the Bielschowsky silver staining in offspring hippocampus. In the CA1 region, nerve axons were much fewer in NP treatment groups com- pared with the control group on both PND21 and PND80 (Fig. 1b). In the CA3 region, axons were smooth and arranged orderly in control group. However, the NP treat- ment groups [especially the 50 and 100 mg/(kg day) groups] presented tortuous and swollen axons, and even the axonal fractures on PND21 and PND80. Meanwhile, more axons were observed in the CA3 region of controls than NP treat- ment pups (Fig. 1b). Together, the above results indicated that developmental exposure to NP resulted in impairments of axons on both PND21 and PND80.
Developmental exposure to NP altered expression of CRMP‑2 and phosphorylated CRMP‑2
in hippocampus
Acting as a major cytoplasmic dynein-interacting com- ponent, CRMP-2 is essential for axonal development by
modulating cytoskeleton (Inagaki et al. 2001; Tan et al. 2015). Hence, we investigated whether developmental exposure to NP had effects on CRMP-2 in offspring hip- pocampus. We observed that positive CRMP-2 staining in hippocampus of pups developmentally exposed to NP was reduced in a dose-dependent manner on both PND21 and PND80 (Fig. 2a, c). Western blot was conducted to further analyze hippocampal expression of CRMP-2 and phospho- rylated CRMP-2 (p-Thr514-CRMP-2, the inactive form of CRMP-2). The results showed that treatment with NP at 50 and 100 mg/(kg day) significantly reduced the expression of CRMP-2 while increased the expression of p-CRMP-2 com- pared to the control group on PND21 and PND80 (Fig. 2b, d; P < 0.05). Treatment with NP at 10 mg/(kg day) tended to reduce the expression of CRMP-2 and increase the expres- sion of p-CRMP-2.
Developmental exposure to NP activated Wnt‑Dvl‑GSK‑3β cascade in hippocampus
CRMP-2 has been identified as a main substrate of GSK-3β in neurons (Yoshimura et al. 2005). Thus, the change in CRMP-2 expression may be due to effects of developmental exposure to NP on Wnt-Dvl-GSK-3β cascade. To determine the underlying mechanisms of NP-induced axonal injury, Wnt-Dvl-GSK-3β cascade was further analyzed. The lev- els of phosphorylated GSK-3β (p-Ser9-GSK-3β, an inactive form of GSK-3β) and total GSK-3β were detected by West- ern blot. The level of p-GSK-3β was significantly decreased by 10, 50, and 100 mg/(kg day) NP treatment on PND21 (Fig. 3a; P < 0.05); however, the level of total GSK-3β did not significantly differ among each group. Dvl and Wnt are believed to act as upstream regulators of GSK-3β cascade (Ciani et al. 2004; Salinas 1999). The levels of Dvl and Wnt were significantly decreased in 50 and 100 mg/(kg day) NP treatment groups compared to the control group (Fig. 3b; P < 0.05). 10 mg/(kg day) NP tended to reduce their lev- els. On PND80, developmental exposure to NP at 50 and 100 mg/(kg day) significantly reduced the level of p-GSK-3β in offspring hippocampus (P < 0.05). Similarly, the total GSK-3β expression remained unchanged compared to the control groups (Fig. 3c). The levels of Dvl and Wnt were significantly decreased by 50 and 100 mg/(kg day) NP treat- ment compared to the control groups (Fig. 3d; P < 0.05). 10 mg/(kg day) NP tended to reduce the levels of p-GSK-3β, Dvl, and Wnt.
NP caused damage to axons in cultured neuronsBecause developmental exposure to NP induced axonal injury in pups in the present study, we further investigated whether NP also impaired axons in cultured neurons. Axons were visualized by immunofluorescence with antibody
Fig. 1 Developmental exposure to NP induced axonal injury in offspring hippocampus. a Rep- resentative images show fluores- cent staining of neurofilament protein 2H3 (green) with DAPI (blue) counterstain on PND21 and PND80 in hippocampus.
b Representative images of Bielschowsky silver staining in the hippocampus on PND21 and PND80 (arrows indicate axons). The images were obtained under the same conditions at
a magnification across each group. Scale bar = 100 μm
Fig. 2 Developmental exposure to NP altered expression of CRMP-2 and phosphoryla-
tion of CRMP-2 in offspring hippocampus. Developmental exposure to NP reduced the positive immunofluorescence staining of CRMP-2 in CA1 and CA3 regions of the hip- pocampus on PND21 (a) and PND80 (c). The images were obtained under the same condi- tions across each group. Scale bar = 100 μm. Developmental exposure to NP decreased the expression of CRMP-2 and increased the expression of
p-CRMP-2 in the hippocampus on PND21 (b) and PND80 (d). The upper bands depict repre- sentative findings for pups from all treatment groups. The lower bar graphs show the results of the semi-quantitative measure- ment of the corresponding protein. Each bar represents the mean ± SEM (n = 6). *P < 0.05
vs. control group, #P < 0.05 vs. 10 mg/kg NP group
Fig. 3 Developmental exposure to NP activated Wnt-Dvl-GSK-3β cascade in offspring hippocampus. Developmental exposure to NP reduced the expression of p-GSK-3β on PND21 (a) and PND80 (c). The expression of total GSK-3β remained unchanged at the differ- ent time points (a, c). The expression of Dvl and Wnt was decreased on PND21 (b) and PND80 (d). The upper bands depict representa-
tive findings for pups from all treatment groups. The lower bar graphs show the results of the semi-quantitative measurement of the cor- responding protein. Each bar represents the mean ± SEM (n = 6).
*P < 0.05 vs. control group, #P < 0.05 vs. 10 mg/kg NP group,
$P < 0.05 vs. 50 mg/kg NP groupfor 2H3. Fluorescent images were captured at a low mag- nification to provide an overall view of cultured neurons (Fig. 4a-I). We found that the average length of neurites was significantly decreased in NP treatment neurons in a dose-dependent manner (Fig. 4a-I, c; P < 0.05). Images of individual neurons were captured at higher magnification for analysis of the longest neurite (axon) (Fig. 4a-II). The length of axons was significantly decreased in NP treatment neurons in a dose-dependent manner (Fig. 4a-II, d; P < 0.05).
Furthermore, we investigated whether NP exposure affected the axonal function of cultured neurons. Active synaptic vesicle recycling, which can be monitored by visualizing the uptake and release of fluorescent dye FM1-43, is a basic feature of axonal function (Ryan et al. 1993). FM1-43 dye was loaded into axons by 45 mM K+ treatment. After excess dye was washed off, the neurons were treated with 90 mM K+ to release the loaded dye from the synaptic vesicles (Fig. 4b, e). We found that the
Fig. 4 NP caused damage to axons in cultured neurons. a Representa- tive images of immunofluorescent staining of neurofilament protein 2H3 (green) with DAPI counterstain (blue) in cultured neurons. b Representative images of loading and unloading of fluorescent dye FM1-43 in cultured neurons. The images were obtained under the same conditions across each group. c Quantification of the length of neurites. d Quantification of the length of the longest neurites (axon).
Data are expressed as mean ± SEM (n = 6). In Fig. 4c, d, *P < 0.05 vs. control group, #P < 0.05 vs. 30 μM NP group, $P < 0.05 vs. 50 μM NP group. e Time-changing trend diagram of fluorescence in loading and unloading of FM1-43. Scale bar = 50 μm. In Fig. 4e, *P < 0.05 vs. 30 μM NP group, #P < 0.05 vs. 50 μM NP group, $P < 0.05 vs. 70 μM NP grouplevel of loaded FM1-43 dye was obviously lower in NP treatment neurons than those in controls. In addition, after 90 mM K+ treatment, the neurons in control group rapidly unloaded FM1-43 within 1 min. FM1-43 dye in control neurons dramatically reduced to quite a low level within 5 min. In contrast, NP treatment (especially at higher con- centrations) compromised the speed and amplitude of the unloading of the fluorescent dye.NP altered expression of CRMP‑2
and phosphorylated CRMP‑2 in cultured neuronsThe expression of CRMP-2 protein was analyzed by immunofluorescence in cultured neurons. We found that the level of CRMP-2 was reduced in cytoplasm and neu- rites of NP treatment neurons in a dose-dependent man- ner (Fig. 5a). Furthermore, the Western blot analysis was
ImageFig. 5 NP altered expression of CRMP-2 and phosphorylation of CRMP-2 in cultured neurons. a Representative images of immunofluorescent staining
of CRMP-2 (red) with DAPI counterstain (blue) in cultured neurons. The images were obtained under the same condi- tions across each group. Scale bar = 100 μm. b Exposure to NP reduced the expression of CRMP-2 and increased expres- sion of p-CRMP-2 in cultured
neurons. The upper bands depict findings for total and phospho- rylated CRMP-2 in cultured neurons from all treatment groups. The lower bar graphs show the results of the semi- quantitative measurement of
the corresponding protein. Each bar represents the mean ± SEM (n = 4). *P < 0.05 vs. control group, #P < 0.05 vs. 30 μM NP group, $P < 0.05 vs. 50 μM NP group conducted to detect the expression of CRMP-2. Similarly, the findings showed that treatment with NP at 30, 50, and 70 μM significantly reduced the expression of CRMP-2 in cultured neurons (Fig. 5b; P < 0.05). 50 and 70 μM NP significantly increased the expression of p-CRMP-2. 30 μM NP also increased the expression, although the difference was not significant.
NP activated Wnt‑Dvl‑GSK‑3β cascade in cultured neurons Our in vivo study indicated that Wnt-Dvl-GSK-3β cas- cade in the offspring hippocampus, which plays a crucial role in axonal remodeling and guidance (Salinas 2005), was activated by NP exposure. Hence, we also investi- gated whether NP exposure affected this signaling path- way in cultured neurons. The findings showed that the level of p-GSK-3β was significantly reduced by 30, 50,
Fig. 6 NP activated Wnt-Dvl-GSK-3β cascade in cultured neurons. a NP exposure reduced the expression of p-GSK-3β in cultured neu- rons. b NP exposure reduced the expression of Dvl and Wnt in cul- tured neurons. The upper bands depict representative findings from
all treatment groups. The lower bar graphs show the results of the semi-quantitative measurement of the corresponding protein. Each bar represents the mean ± SEM (n = 4). *P < 0.05 vs. control group, #P < 0.05 vs. 30 μM NP group, $P < 0.05 vs. 50 μM NP groupand 70 μM NP treatment (Fig. 6a; P < 0.05), whereas the level of total GSK-3β was not affected (Fig. 6a). Further- more, the expression of Dvl was significantly reduced by 50 and 70 μM NP treatment, and the expression of Wnt was significantly reduced by 30, 50, and 70 μM NP treatment (Fig. 6b; P < 0.05). These data indicated that NP treatment also activated Wnt-Dvl-GSK-3β cascade in cultured neurons.
SB attenuated NP‑induced axonal injury in cultured neurons
To further clarify the role of Wnt-Dvl-GSK-3β cascade in NP-induced axonal injury, SB, a specific GSK-3β inhibi- tor (Shen et al. 2011), was employed in this study. Our data showed that treatment of neurons with SB and NP significantly increased the average length of neurites than treatment with NP alone (Fig. 7a-I, c; P < 0.05). Similarly, treatment of neurons with SB and NP caused a significant increase in the length of the longest neurites (axons) than
ImageFig. 7 SB attenuated NP- induced axonal injury. a Representative images of immunofluorescent staining of neurofilament protein 2H3
(green) with DAPI counterstain (blue) in cultured neurons.
b Representative images of loading and unloading of fluorescent dye FM1-43 in cultured neurons. The images were obtained under the same conditions across each group. c Quantification of the length of neurites. d Quantification of the length of the longest neurites (axon). Data are expressed as mean ± SEM (n = 6). In Fig. 7c, d, *P < 0.05 vs. control group,
#P < 0.05 vs. 50 μM NP group. e Time-changing trend diagram of fluorescence in loading
and unloading of FM1-43. In Fig. 7e, *P < 0.05 vs. control group, #P < 0.05 vs. 50 μM NP + SB group, $P < 0.05 vs.
SB group. f Co-treatment SB and NP significantly increased CRMP-2 expression and decreased p-CRMP-2 expres- sion compared to 50 μM NP treatment group. The upper bands depict representative find- ings from all treatment groups. The lower bar graphs show the results of the semi-quantitative measurement of the correspond- ing protein. Each bar represents the mean ± SEM (n = 4). In
Fig. 7f, *P < 0.05 vs. control group, #P < 0.05 vs. 50 μM NP group. Scale bar = 50 μmtreatment with NP alone (Fig. 7a-II, d; P < 0.05). In addition, the FM1-43 staining experiment showed that co-treatment of SB and NP significantly increased the level of loaded FM1- 43 dye in neurons than those exposed to NP alone (Fig. 7b, e; P < 0.05). Neurons subjected to treatment of SB and NP also showed significantly increased speed and amplitude of the unloading of the fluorescent dye (Fig. 7b, e; P < 0.05) than those exposed to NP alone. Meanwhile, we also deter- mined the expression of CRMP-2 and p-CRMP-2 in cul- tured neurons. In line with expectations, their co-treatment in neurons caused a significant increase in CRMP-2 and a significant decrease in p-CRMP-2 than treatment with NP alone (Fig. 7f; P < 0.05).
Discussion
It has been well known that certain periods during develop- ment, such as prenatal period and early postnatal life, are critically sensitive windows of exposure to some chemicals (Walker and Gore 2017). Although the placenta may provide protective defenses against harmful exposures, many lipo- philic chemicals, such as NP, can enter into umbilical cord blood via the placental barrier (PB) (Grandjean and Landri- gan 2006). Furthermore, during these periods, blood–brain barrier (BBB) has not been formed completely, which makes the developing brain more vulnerable to chemicals. Recently, it has been shown that developmental NP expo- sure causes neurotoxicity and impairs learning and memory capacity (Couderc et al. 2014; Gu et al. 2018; Jie et al. 2010, 2016, 2017). Since axon is the vital structure of the neural networks for brain function and the most unique and primary component of neuron (Kennedy 2013), the effects of devel- opmental exposure to NP on axon are urgently needed to be studied. Thus, in the present study, we established the ani- mal model that developmentally exposed to NP by maternal gavage during perinatal period, and evaluated the effects of NP exposure on axonal growth. In addition, to further study NP-induced axonal injury and the underlying mechanisms, primary cultured neurons were also directly exposed to NP. Bevan et al. found that NP treatment impaired NGF- induced neurite outgrowth both in rat pheochromocytoma (PC12) cell and embryonic Xenopus spinal cord neurons, which indicated adverse impacts of NP exposure on early neural development (Bevan et al. 2006). However, little is known regarding the effect of developmental NP exposure on axon. In the present study, Bielschowsky sliver stain- ing was conducted to assess the damage to the axon caused by developmental exposure to NP, which indicated axon loss and axonal fracture in the hippocampus of treated pups. Immunofluorescence staining of neurofilament pro- tein 2H3 showed that NP treatment resulted in decreases in axonal length and density. These data support the idea
that developmental exposure to NP causes the injury of axon. Particularly intriguing was that the observation of the impaired axon was on PND80, in addition to PND21, when the exposure to NP had already been terminated. This may suggest that the injury of axon caused by developmental exposure to NP is largely irreversible.
CRMP-2 is exclusively expressed in the growing axon of neurons, and widely used as a major indicator for axonal growth (Fukata et al. 2002; Inagaki et al. 2001). In this study, developmental exposure to NP significantly reduced the expression of CRMP-2 and increased the expression of p-CRMP-2 in offspring. CRMP-2 induces the forma- tion of axon, while p-CRMP-2 loses the ability of microtu- bule assembly, and thereby inhibits the formation of axon (Cole et al. 2004). Thus, this result is in line with the data of Bielschowsky sliver staining and neurofilament protein 2H3. Moreover, the alteration in the expression of CRMP-2 and p-CRMP-2 may underlie the damage to axon.
GSK-3β plays a pivotal role in cytoskeletal organi- zation and neuroplasticity (Salcedo-Tello et al. 2011; Yoshimura et al. 2006). Specifically, the inactivation of GSK-3β is necessary for axonal formation, and phospho- rylation at Ser 9 is a major way to inactivate GSK-3β (Jiang et al. 2005). Tiwari et al. found that developmen- tal exposure to bisphenol A (BPA) decreased the expres- sion of p-Ser9-GSK-3β in the rat hippocampus (Tiwari et al. 2016). Tetrachlorodibenzo-p-dioxin (TCDD) expo- sure has also been shown to decrease the expression of p-Ser9-GSK-3β in the brain cortex of adult rat (Xu et al. 2013). In the current study, developmental exposure to NP decreased the phosphorylation of GSK-3β at Ser9, which indicates the activation of GSK-3β. CRMP-2 is an important downstream target of GSK-3β. Thus, these results may provide evidence that the activation of GSK-3β observed in the hippocampus of NP-exposed pups was responsible for the alteration in phosphorylation of CRMP-2, and consequently impaired the development of axon. GSK-3β is a downstream member of Wnt-Dvl- GSK-3β cascade, which is a crucial signaling for axonal growth (Ciani and Salinas 2007). Wnt-7a, broadly distrib- uted in hippocampal neurons, regulates axonal growth by modulating downstream signaling events, and one of the most important them is the deactivation of GSK-3β (Sali- nas 1999). DVL is a key scaffolding protein and mediator in Wnt signaling pathway (Kim et al. 2018). It is closely associated with the regulation of axonal microtubules through inhibition of GSK-3β (Krylova et al. 2000). As expected, both the levels of Dvl and Wnt were decreased in the hippocampus of pups exposed to NP in the present study. The decreased levels of Dvl and Wnt may account for the activation of GSK-3β. Furthermore, it is reason- able to speculate that the disturbance of Wnt-Dvl-GSK-3β cascade may be involved in impairments of axons induceby developmental exposure to NP. To clearly summarize the involvement of Wnt-Dvl-GSK-3β cascade in NP- induced axonal injury, a schematic diagram is showed in supplementary Fig. 1.
Our result also showed that even months after the treat- ment, the disturbance of the Wnt-Dvl-GSK-3β cascade was not fully restored. It has been shown that exposure to certain POPs at the critically developmental period of brain permanently alters some signaling in the CNS (Grandjean and Landrigan 2006; Hu et al. 2014; Men- nigen et al. 2018; Tiwari et al. 2016). The irreversible disturbance of the Wnt-Dvl-GSK-3β cascade observed in this study induced by developmental exposure to NP is in accordance with these alterations.
To further investigate NP-induced axonal injury and the underlying mechanisms, primary cultured neurons were also exposed to NP. The in vitro study allows us to determine the active synaptic vesicle recycling in axons by FM1-43 staining experiment. NP exposure led to the alteration in uptake and release of fluorescent dye FM1-43 in neurons, which indicated that synaptic vesicle recycling was damaged in NP-treated neurons. As a basic feature of axonal function, the suppressed synaptic vesi- cle recycling indicated that NP exposure affected axonal function of cultured neurons. The immunofluorescence assay of neurofilament protein 2H3 was also conducted in cultured neurons. Similar to the in vivo results, the data indicated that NP exposure decreased the length of axon. Together, NP treatment damaged the development of axon both in offspring hippocampus and in primary cultured neurons.
NP exposure also reduced the expression of CRMP-2 in cultured neurons, while increased the expression of p-CRMP-2, consistent with the in vivo observation. Fur- thermore, NP exposure was found to reduce the phospho- rylation of GSK-3β at Ser9, and the levels of Wnt and Dvl in cultured neurons. Considering the importance of Wnt-Dvl-GSK-3β cascade in axonal development, these data suggested that NP exposure over-activated the Wnt- Dvl-GSK-3β cascade in cultured neurons and impaired the axonal development.
SB, a small molecule WNT mimetic, is often used to inhibit the activation of GSK-3β (Shen et al. 2011). To confirm the contribution of activated Wnt-Dvl-GSK-3β cascade to the axonal damage caused by NP exposure, we used it as a tool to deactivate this cascade in the NP- exposed neurons. In this study, SB application recovered the shortening of the average length of axon induced by NP. It also attenuated NP-induced damage to axonal func- tion. Moreover, SB reversed the increase of p-CRMP-2 and the decrease of CRMP-2. These data supported the idea that the activation of Wnt-Dvl-GSK-3β cascade contrib- uted to impairment of axonal development induced by NP.
Conclusions
In conclusion, our results showed that developmental exposure to NP led to an obvious decrease in axonal length and density in the hippocampus of pups. Developmental exposure to NP also altered the expression of CRMP-2 and p-CRMP-2 in the hippocampus, and over-activated Wnt-Dvl-GSK-3β cascade, the upstream signaling of CRMP-2 that regulates axonal development. Even months after developmental exposure to NP, impairment of axonal development and alteration of this cascade in the offspring hippocampus were not fully restored. Primary cultured neurons were also exposed to NP, and decreased axonal length and impaired axonal function caused by NP were observed. Similar to the in vivo results, NP also altered expression of CRMP-2 and p-CRMP-2 and activated Wnt-Dvl-GSK-3β cascade. SB recovered the shortening of axons and the impairment of axonal function induced by NP. Collectively, our results support the hypothesis that exposure to NP impairs axonal development. Furthermore, our findings also demonstrate that the activation of Wnt- Dvl-GSK-3β cascade contributes to impairment of axonal development induced by NP.
Acknowledgements This work was supported by Natural Science Foundation of Liaoning Province, China (Grant number: 201602865), Program for Liaoning Innovative Research Team in University (Grant number: LT2015028), and Liaoning Revitalization Talents Program (Grant number: XLYC1807225).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval All applicable international, national, and/or institu- tional guidelines for the care and use of animals were followed.
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