Neurodevelopmental disorders can be caused by exposure to environmental toxicants resulting in abnormal neuronal proliferation and/or differentiation. Valproic acid (VPA) is a clinically used mood stabilizer and antiepileptic drug. VPA has been suggested as a teratogen that can induce neurodevelopmental disorders, such as fetal valproate syndrome and autism spectrum disorders (Dalens
The T-type calcium channel is a low-voltage activated channel that regulates calcium-dependent physiological processes at the resting membrane potential in excitable cells or non-excitable cells, such as embryonic progenitor cells (NPCs) (Iftinca and Zamponi, 2009; Louhivuori
In this study, we investigated whether genes encoding T-type calcium channels are epigenetic targets during prenatal exposure to VPA and assessed the role of T-type calcium channels in VPA-induced neurodevelopmental abnormalities. We found that the mRNA levels of T-type calcium channels were increased by VPA treatment. CaV3.1 was identified as the epigenetic target of VPA, as its increase was not detected in NPCs treated with valpromide, a carboxamide derivative of VPA that does not inhibit histone deacetylase. The proliferation of VPA was increased in NPCs, and pharmacological blockade of T-type calcium channels prevented this increased proliferation. Cells that differentiated from NPCs treated with VPA exhibited abnormal calcium ion (Ca2+) response to KCl-induced depolarization stimulation. Our results provide a clue for understanding the role of T-type calcium channels in the pathophysiology of neurodevelopmental disorders.
Pregnant female Sprague-Dawley rats were obtained from Orient Bio (Gyeonggi-do, Korea). Animal housing and treatments, including anesthesia, euthanasia, and administration of VPA, were carried out in accordance with the principles of laboratory animal care (NIH Publication No. 85-23, revised 1985) and were approved by the animal care and use committee of Konkuk University, Korea (KU14143).
VPA was administered as previously reported (Kim
Primary NPCs were isolated from the E14.5 cortices obtained from the rat embryos and were maintained in a humidified chamber at 37°C with growth factors, as described previously (Go
Total RNA was extracted from NPCs or cortices using TRIzol reagent (Invitrogen). The RNA was reverse transcribed using a RevertAid Reverse transcriptase kit (K1622; Fermentas, Waltham, MA, USA). For quantitative real-time PCR, cDNA was amplified with custom-made primers using SYBR® Premix Ex Taq II (RR820A; TaKaRa Bio, Shiga, Japan) in an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Fold-changes in gene expression were quantified using the comparative threshold cycle (Ct) method. The primer sequences were as follows:
Tissues and cells were lysed with 2× sample buffer (4% w/v sodium dodecyl sulfate [SDS], 20% glycerol, 200 mM dithiothreitol [DTT], 0.1 M Tris-HCl, pH 6.8, and 0.02% bromophenol blue), including protease and phosphatase inhibitors. Protein samples were quantified using a standard bicinchoninic acid (BCA) analysis and an equal amount of total protein was resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 1% skim milk and incubated overnight at 4°C with primary antibodies against CACNA1G, (ab134269; Abcam, Cambridge, UK; 1:2,000), β-actin (A2066; Sigma-Aldrich; 1:50,000), Histone H3 (9715S; Cell Signaling Technology, Beverly, MA, USA; 1:2,500), or acetylated Histone H3 (06-599; Millipore, Billerica, MA, USA; 1:2,500). After washing each membrane three times with 0.1% Tris-buffered saline (TBS-Tween), the blots were incubated with the peroxidase-conjugated secondary antibody for 2 h at 20-25°C. After washing the blots with 0.1% TBS-Tween, bands were detected using Amersham enhanced chemiluminescence reagent (GE Healthcare Life Science, Pittsburgh, PA, USA) and visualized using a LAS-3000 imaging system (Fuji Film, Tokyo, Japan). The band intensity was analyzed using the Multi Gauge v3.0 software (Fuji Film).
Chromatin immunoprecipitation was performed as previously reported (Kim
NPCs were plated on coverslips and fixed with 4% paraformaldehyde at 4°C for 15 min. Samples were permeabilized with 0.1% Triton X-100 dissolved in blocking buffer for 15 min and blocked with blocking buffer comprising 1% bovine serum albumin and 3% fetal bovine serum in PBS for 30 min at 20-25°C temperature. Coverslips were incubated overnight at 4°C with primary antibodies. After three washes, the coverslips were incubated with secondary antibodies conjugated with donkey anti-mouse or donkey anti-rabbit IgG for 1 h at 20-25°C temperature. The coverslips were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA), and cells were imaged using a model Bx61 fluorescence microscope (Olympus, Tokyo, Japan).
Calcium imaging was carried out in NPCs plated on coverslips. Tyrode’s solution contained 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, and 30 mM glucose (pH adjusted to 7.4). To record calcium entry, coverslips were incubated with 5 mM Fluo3-AM (Invitrogen) in Tyrode’s solution supplemented with 0.02% Pluronic F-127 (Sigma-Aldrich) for 30 min at 37°C in the dark. The coverslips were placed in a perfusion chamber on a model Ti2 inverted microscope (Nikon, Tokyo, Japan). Calcium was visualized with a filter set with an excitation peak of 480 nm, 490 nm long pass mirror, 500-550 nm emission filter, and manual flip shutter. Images were captured every 1 s using a model DS-Qi2 camera (Nikon). For depolarization, 15 and 50 mM KCl were added to the perfusion chamber. Fmax and Fbase denote maximal increase after stimulation and baseline fluorescence, respectively. Fluorescence change (ΔF) was normalized using the following equation: ΔF=(Fmax–Fbase)/Fbase. Calcium responsive cells displayed a ΔF after depolarization>3 times the baseline Fbase.
Cell viability was measured using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based assay. After 24 h of drug treatment, MTT (Sigma-Aldrich, 200 μg/mL) was added to each well and incubated for 2 h at 37°C. After removing the medium, dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals formed by viable cells. Absorbance was measured using a microplate reader at a wavelength of 570 nm and a reference filter of 620 nm, using a SpectrMax190 device (Molecular Devices, Sunnyvale, CA, USA).
Statistical analyses were performed using Prism software (GraphPad, La Jolla, CA, USA). Data are presented as mean ± SEM. After confirming the normality of data in datasets, Student’s
Previously, we showed that VPA induces abnormal genetic transcriptional activation by inhibiting histone deacetylase in the embryonic cortex, which results in neurodevelopmental defects (Kim
NPCs are proliferative cells with the potential to differentiate into different types of neuronal cells. In our previous study, we showed that VPA induces abnormal differentiation and proliferation of NPCs, which results in impaired embryonic cortical development (Go
To investigate whether VPA promotes CaV3.1 expression through histone modification, we treated NPCs with VPM (0.5 mM), which is a derivative of valproic acid that does not inhibit histone deacetylase. As expected, VPA significantly increased CaV3.1 expression but VPM did not (Fig. 3A). Next, to confirm that the increase in CaV3.1 protein is induced through epigenetic modulation, we treated NPCs with VPA, VPM, and two additional histone deacetylase inhibitors, trichostatin A (0.2 M) and sodium butyrate (0.1 mM). The levels of CaV3.1 were significantly increased by VPA, trichostatin A, and sodium butyrate, but not by VPM (Fig. 3B).
Next, we examined the binding of acetylated histone H3 to promoter regions of the
We previously showed that VPA facilitates proliferation and promotes Pax6 expression in the embryonic cortex and NPCs, which results in macrocephaly and excitatory-inhibitory imbalance, respectively, in an animal model of autism induced by prenatal exposure to VPA (Go
Next, we questioned whether activity-dependent calcium influx is altered in differentiated neural cells from VPA-treated NPCs. We measured the calcium response to KCl-induced depolarization stimulation using Fluo3-AM in the differentiated neural cells from NPCs treated with either vehicle or VPA 24 hrs prior. Differentiated neural cells from VPA-treated NPCs displayed an increase in calcium influx in response to 15 mM KCl-induced depolarization stimulation, compared to differentiated neural cells from vehicle-treated NPCs (Fig. 6A). Calcium responsive cells, in response to both 15 and 50 mM KCl stimulation, were also increased by VPA treatment (Fig. 6B). To confirm whether the increased activity-dependent Ca2+ influx was induced by increased levels of L-type calcium channels, we measured the levels of
In this study, we found that VPA increases the mRNA levels of all subtypes of T-type calcium channels in primary cultured rat NPCs and embryonic rat cortex. CaV3.1 protein levels were increased by other histone deacetylase inhibitors, but not by VPM, suggesting that CaV3.1 is an epigenetic target of VPA during embryonic cortical development. Pharmacological blockade of T-type calcium channels prevented an increase in VPA-induced proliferation of NPCs. Lastly, we found that activity-dependent Ca2+ influx is increased in differentiated neural cells from NPCs previously exposed to VPA. Our results may provide a clue for understanding the role of T-type calcium channels in VPA-induced neurodevelopmental impairments.
We found that T-type calcium channels are upregulated by VPA exposure during the embryonic period. T-type calcium channels have been implicated in neurodevelopmental disorders, such as autism spectrum disorders (Splawski
We found that VPA increased the number of viable NPCs, indicating that VPA increased the proliferation of NPCs. Of note, the increased proliferation of NPCs exposed to VPA was prevented by T-type calcium channel blockers, suggesting a crucial role of T-type calcium channels in mediating the abnormal proliferation of NPCs exposed to VPA. Indeed, the importance of T-type calcium channels in regulating the cell cycle and cell proliferation has been reported in proliferating cells, such as cancer cells (Hirooka
Differentiated neural cells from VPA-treated NPCs showed increased Ca2+ influx in response to KCl-induced depolarization, suggesting that prenatal exposure to VPA causes an abnormality in activity-dependent Ca2+ entry. Given VPA promotes differentiation into excitatory neurons by upregulating Pax6 levels (Kim
In conclusion, our study suggests that up-regulation of T-type calcium channels mediates the abnormal proliferation in VPA-exposed NPCs, which might lead to neurodevelopmental disorders. Additionally, abnormal activity-dependent Ca2+ entry may also contribute to the pathophysiology of VPA-induced neurodevelopmental disorders. How Ca2+ derived from T-type calcium channels is involved in the proliferation of NPCs and how abnormal activity-dependent Ca2+ signaling affects the pathophysiology of VPA-induced neurodevelopmental disorders require further investigation.
This research was supported by the Chung-Ang University Research Grants in 2019 and by the Korea Institute of Science and Technology (Grant No. 2E30190-20-060).