Edited by: Cristina Cereda, IRCCS National Neurological Institute “C. Mondino”, Italy
Reviewed by: Mariano Soiza-Reilly, CONICET Institute of Physiology, Molecular Biology and Neurosciences (IFIBYNE), Argentina; Adelaide Fernandes, University of Lisbon, Portugal
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Selective serotonin reuptake inhibitors (SSRIs) are frequently used to treat depression during pregnancy. Various concerns have been raised about the possible effects of these drugs on fetal development. Current developmental neurotoxicity (DNT) testing conducted in rodents is expensive, time-consuming, and does not necessarily represent human pathophysiology. A human,
Between 7 and 12% of pregnant women suffer from depression (Bennett et al.,
Paroxetine was shown to cross the placental barrier (Hendrick et al.,
The use of paroxetine in pregnancy has declined substantially (Meunier et al.,
DNT is of high concern, however, no routine testing for DNT is carried out in any regulatory program worldwide. Indeed, DNT testing is not required unless triggered by the observation of neurotoxic or endocrine effects in adult rodents. Furthermore, as described in the OECD guidelines, DNT experiments are also extremely expensive (1.4 million per substance), as well as time- and animal-consuming (1,400 pups per compound). Moreover, human brain complexity may not be completely reflected in animal models. The same shortcomings apply for toxicity testing of drugs developed in the pharmaceutical industry. Thus, thousands of drugs and chemicals reach the market without proper classification regarding DNT.
There is consensus in the field that more reliable and efficient screening and assessment tools are required for better identification and evaluation of DNT chemicals and drugs. Over the last 15 years, there has been a process to develop an
The previously described 3D human iPSC-derived brain model (BrainSpheres) recapitulates some of the key events of neurodevelopment (Pamies et al.,
Paroxetine was supplied by Sigma. A stock of 10 μg/ml was prepared in DMSO Hybri-Max (Sigma) and stored at −20°C. DMSO (0.072%) was used as vehicle control to match the amount of DMSO in the highest paroxetine concentration of 60 ng/ml.
The CRL-2097 line was derived from CCD-1079Sk ATCC® CRL-2097™ fibroblasts purchased from ATCC and was kindly provided by Dr. Hongjun Song within our joint NIH NCATS funded project (Pamies et al.,
Cytotoxicity to BrainSpheres was assessed after exposure to 0, 20 and 60 ng/ml of paroxetine continuously for 8 weeks. After drug exposure, resazurin reduction assay was performed. One-hunderd microliter of 2 mg/ml Resazurin were added directly to 6-well plates (2 ml/well). The plates were incubated for 3 h at 37°C, 5% CO2. Subsequently, 50 μl of medium from each well were transferred to 96-well plates and the fluorescence of resorufin was measured at 530 nm/590 nm (excitation/emission) using a multi-well fluorometric reader CytoFluor series 4000 (PerSeptive Biosystems, Inc., Framingham, MA, USA). To determine statistical significance, an one-way ANOVA test was performed with
Mitochondrial dysfunction was measured by MitoTracker Red CMXRos (Life Technologies, Carlsbad, CA, USA) following the protocol described in Pamies et al. (
BrainSpheres were collected at 8 weeks of differentiation, washed three times for 5 min with PBS and fixed with 4% PFA for 1 h at room temperature followed by two washing steps with PBS. BrainSpheres were incubated for 1 h in blocking solution consisting of 5% normal goat serum (NGS) in PBS with 0.4% Triton-X100 (Sigma). BrainSpheres were then incubated for 48 h at 4°C with a combination of primary antibodies (
Primary antibodies for immunohistochemistry.
Antibody | Host | Type | Source | Dilution |
---|---|---|---|---|
NF200 | Rabbit | Polyclonal | Sigma | 1:200 |
GFAP | Rabbit | Polyclonal | Dako | 1:500 |
βTUBIII | Rabbit | Polyclonal | R&D | 1:200 |
S100β | Rabbit | Polyclonal | Santa Cruz | 1:200 |
O4 | Mouse | Monoclonal | R&D | 1:200 |
O1 | Mouse | Monoclonal | Millipore | 1:500 |
MBP | Mouse | Monoclonal | Biolegend | 1:200 |
Synaptophysin | Mouse | Monoclonal | Sigma | 1:200 |
PSD95 | Rabbit | Monoclonal | Life technologies | 1:200 |
Secondary antibodies for immunohistochemistry | ||||
Alexa Fluor 488 goat anti-mouse | Life Technologies | 1:500 | ||
Alexa Fluor 658 goat anti-rabbit | Life Technologies | 1:500 |
After 8 weeks of differentiation, BrainSpheres were fixed and stained for synaptophysin (SYP, pre-synaptic protein;
BrainSpheres were collected after paroxetine exposure for 8 weeks. One-hundred and fifty micro liter RIPA lysis buffer (Sigma–Adrich, St. Louis, MO, USA) containing 1× protease inhibitor cocktails (Sigma–Adrich, St. Louis, MO, USA) was added to each sample and incubated on ice for 30 min to lyse the cells. Then the samples were centrifuged at 12,000 rpm, 4°C for 15 min, the supernatant was transferred to a new tube and incubated with pierce™ lance marker reducing sample buffer (Thermo Fisher Scientific, Waltham, MA, USA) at 95°C for 5 min. The protein concentration was measured with the pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). A total of 30 μg protein was separated on a 4–15% gradient SDS-polyacrylamide gel with 80 V for 120 min and transferred to a polyvinylidene difluoride membrane by electroblotting with 200 mA on ice for 2 h. The non-specific membrane binding was blocked with a blocking solution (PBS, 0.5% Tween-20, 5% non-fat dry milk, pH 7.4) for 1 h at room temperature. Subsequently, the membrane was incubated with primary antibodies Synaptophysin, 1:800, Sigma–Adrich, St. Louis, MO, USA; PSD95, 1:1,000, Thermo Fisher Scientific, Waltham, MA, USA; GAPDH, 1:1,000, Cell Signaling Technology, Danvers, MA, USA) in blocking solution overnight at 4°C. The membrane was washed thoroughly with PBS-T and incubated with HRP-conjugated goat anti-mouse or goat-anti-rabbit secondary antibodies anti-mouse, 1:3,000, BIO-RAD; anti-rabbit, 1:2,000, Cell Signaling Technology, Danvers, MA, USA)in blocking solution at room temperature for 1 h. The blotting bands were detected by chemiluminescence reagent plus (BIO-RAD, Hercules, CA, USA), and exposed to the X-ray film.
BrainSpheres were cultivated as described above. After 8 weeks of exposure to 0, 20 or 60 ng/ml of paroxetine, BrainSpheres were seeded on Matrigel™ (BD Biosciences) pre-coated, flat-bottom, black 24-well plates (Thermo Fisher Scientific, Waltham, MA, USA). After 24 h, the BrainSpheres were fixed in 4% PFA, stained with anti-β-III-Tubulin (neuronal marker) and GFAP (astrocytes marker) as described above and imaged using a confocal microscope Zeiss UV-LSM 510 and analyzed using the Sholl ImageJ Software
BrainSpheres were exposed for 8 weeks to 0, 20 or 60 ng/ml of Paroxetine, fixed with 4% PFA and stained with anti-O4 antibody. Immunohistochemistry was performed as described above. O4-positive cells were counted in four different experiments by four different individuals, median and standard deviation (SD) were calculated from the count of each individual.
To determine, if therapeutic concentrations could produce general cytotoxicity, resazurin assay, and mitotracker analysis were performed after 8 weeks of exposure to paroxetine (0, 20 and 60 ng/ml) in two cell lines (
Cell viability, mitochondria function and immunohistochemistry of BS after exposure to paroxetine. BrainSpheres were treated during the differentiation process (8 weeks) with 20 or 60 ng/ml paroxetine. This is a representative figure of the experiment performed, both cell lines have shown similar results.
BrainSpheres were exposed to therapeutic-relevant paroxetine concentrations (Tomita et al.,
Synaptic markers analysis after paroxetine exposure. BrainSpheres were exposed to paroxetine (0, 20 or 60 ng/ml) for 8 weeks of differentiation. After 8 weeks BrainSpheres were collected to perform immunohistochemistry and Western blot.
BrainSpheres were cultivated for 8 weeks with and without the presence of paroxetine (20 or 60 ng/ml). In order to quantify neurite outgrowth, BrainSpheres were attached to Matrigel-coated 24-well plates after 8 weeks of exposure to paroxetine and cultured for further 24 h. Neurite outgrowth analysis showed a consistent statistically significant decrease in neurite density in both cell lines treated with 60 ng/ml of paroxetine in different experiments (
Neurite outgrowth analysis after paroxetine exposure. BrainSpheres were cultivated for 8 weeks with and without paroxetine (20 or 60 ng/ml). After treatment, cells were seeded on Matrigel® for 24 h and fixed for immunohistochemistry.
BrainSpheres were cultivated for 8 weeks in the presence and absence of paroxetine (0, 20 or 60 ng/ml). After 8 weeks of treatment, BrainSpheres were collected, fixed and stained with different antibodies as described in materials and methods (
Quantification of oligodendrocytes population. BrainSpheres were cultivated for 8 weeks with and without the presence of paroxetine (0, 20 or 60 ng/ml). After treatment, spheres were fixed for immunohistochemistry.
Paroxetine, a SSRI is contraindicated during the first trimester of pregnancy mainly because of the increased risk of cardiac and other congenital malformations (Cole et al.,
First and early second trimesters of pregnancy are vital for the development of the heart (Mäkikallio et al.,
Serotonin plays an important role in cognitive processes, such as memory and learning (Berridge et al.,
Effects of paroxetine on key processes of brain development have to be established in order to evaluate its potential DNT. However, current DNT testing is facing numerous challenges. DNT experts have raised concerns about the relevance of animal data for human risk assessment and have recommended substituting the expensive and time-consuming rodent guidelines for an
BrainSpheres exposed to 20 or 60 ng/ml paroxetine for 8 weeks did not present any cytotoxic effects or mitochondrial dysfunction (
Gene expression analysis for the serotonin transporter (SLC6A4;
We also observed a statistically significant decrease in neurite outgrowth at 60 ng/ml in all the experiments with both lines, however, 20 ng/ml presented differing results (
Oligodendrocyte differentiation and myelin formation are two key events of neural development that have remained difficult to cover in DNT test batteries due to the difficulty to differentiate oligodendrocytes
In conclusion, some indications from clinical studies suggested that paroxetine may affect brain development, but these results were inconsistent. By using a battery of assays that cover several key events of neural development in BrainSpheres we were able to detect alterations in neurite outgrowth, reduction of synaptic marker expression and a decrease in the number of oligodendrocytes after exposure to paroxetine at relevant therapeutic concentrations. These results identify paroxetine as a potential human developmental neurotoxicant, and suggest that the contraindication for its use should be evaluated and possibly extended far beyond the first trimester of pregnancy. In addition, we show that BrainSpheres allow to cover different aspects of brain development in one single system and constitute a novel tool to study and identify potential developmental neurotoxicants among chemicals and drugs, before their entry to the market.
The datasets generated for this study are available on request to the corresponding author.
XZ: western blots, stainings, and some cultures. GH: neurite outgrowth. LS: neurite outgrowth, immunohistochemistry, and supervision XZ, VZ, and MC: myelin and oligodendrocytes quantification. M-GZ: oligodendrocytes quantification, writing and revision of the manuscript. RS: BS cultures, viability assays, and mitochondrial function. FB and PB: synapsis quantification. PB: Synapsis quantification. HH: revision of the manuscript. TH: project idea, PI funding, head of laboratory, and revision of the manuscript. DP: cultures, immunohistochemistry, neurite outgrowth analysis, statistical analysis, coordinator of the experiments, and writer of the manuscript.
TH, HH and DP are named inventors on a patent by Johns Hopkins University on the production of mini-brains, which is licensed to AxoSim, New Orleans, LA, USA. They consult AxoSim and TH is shareholder. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
BrainSpheres oligodendrocytes quantification panel. Confocal imagines of O4 positive marker for the four different experiments used for quantification.
Myelin quantification assay. BrainSphere myelination was quantified using a protocol adapted from Kerman et al. (
Gene expression of SLC6A4. BrainSphere gene expression of SLC6A4 was quantified using qPCR. Housekeeping was Actin. No statistically significant changes were found.
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