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Exp Neurobiol 2017; 26(5): 252-265
Published online October 31, 2017
© The Korean Society for Brain and Neural Sciences
Ki Chan Kim1, Chang Soon Choi1, Edson Luck T. Gonzales1, Darine Froy N. Mabunga1, Sung Hoon Lee2, Se Jin Jeon3, Ram Hwangbo4, Minha Hong5, Jong Hoon Ryu6, Seol-Heui Han1, Geon Ho Bahn7* and Chan Young Shin1*
1School of Medicine and Center for Neuroscience Research, SMART Institute of Advanced Biomedical Sciences, KU Open Innovation Center, Konkuk University, Seoul 05029, 2College of Pharmacy, Chung-Ang Univeristy, Seoul 06974, 3Center for Neuroscience, Korea Institute of Science and Technology, Seoul 02792 , 4Department of Psychiatry, Kyung Hee University Hospital, Seoul 02447, 5Department of Psychiatry, Seonam University, College of Medicine, Myongji Hospital, Goyang 10475, 6Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 7Department of Neuropsychiatry, School of Medicine, Kyung Hee University, Seoul 02447, Korea
Correspondence to: *To whom correspondence should be addressed.
Chan Young Shin, TEL: 82-2-2030-7834, FAX: 82-2-2049-6192, e-mail: firstname.lastname@example.org
Geon Ho Bahn, TEL: 82-2-958-8556, FAX: 82-2-969-6958, e-mail: email@example.com
The valproic acid (VPA)-induced animal model is one of the most widely utilized environmental risk factor models of autism. Autism spectrum disorder (ASD) remains an insurmountable challenge among neurodevelopmental disorders due to its heterogeneity, unresolved pathological pathways and lack of treatment. We previously reported that VPA-exposed rats and cultured rat primary neurons have increased Pax6 expression during post-midterm embryonic development which led to the sequential upregulation of glutamatergic neuronal markers. In this study, we provide experimental evidence that telomerase reverse transcriptase (TERT), a protein component of ribonucleoproteins complex of telomerase, is involved in the abnormal components caused by VPA in addition to Pax6 and its downstream signals. In embryonic rat brains and cultured rat primary neural progenitor cells (NPCs), VPA induced the increased expression of TERT as revealed by Western blot, RT-PCR, and immunostainings. The HDAC inhibitor property of VPA is responsible for the TERT upregulation. Chromatin immunoprecipitation revealed that VPA increased the histone acetylation but blocked the HDAC1 binding to both
Keywords: telomerase reverse transcriptase, valproic acid, autism, excitatory/inhibitory imbalance, glutamatergic neuronal differentiation
The prenatally VPA-exposed rodent is a valid environmental factor animal model of autism spectrum disorder (ASD) widely utilized for its clinical relevance. However, despite the substantial studies conducted using this animal model, the factors and pathways that can delineate the pathophysiology of ASD are still unknown. A druggable target must be determined to effectively carry out the investigations involving this model and to help devise a treatment for the heterogeneous and seemingly insurmountable ASD. Previously, we have elucidated that VPA affects the glutamatergic differentiation of prenatal rat brains through Pax6 , with a possible involvement of other components, which can be essential for characterizing the mechanisms of ASD.
Telomeres are regions of DNA repeat sequences located at each end of a chromosome . During cell division, the enzymes duplicating DNA are unable to continue their duplication until the chromosomes' ends . As a result, the length of telomeres is decreased by the division of cell . In lively dividing cells, however, decreased length of telomeres is constantly restored by telomerase. Telomerase is an RNA-dependent polymerase which lengthens telomeres in DNA strands by adding TTAGGG telomere repeat sequence to the chromosome's ends. This addition prevents shortening of the chromosomal ends even after multiple rounds of replication. Telomerase is composed of two protein components including reverse transcriptase activity (TERT, telomerase reverse transcriptase) and an RNA template (TERC, telomerase RNA component) [5,6]. The activity and expression of telomerase in neural progenitor cells (NPCs) are reported as relatively high, and declines when NPCs differentiate to neuronal fates. There is a decrease in the RNA component of TERT observed before the initiation of neuronal differentiation while the expression level of TERT is constantly elevated during brain development in mice, hinting an involvement of telomerase activity in nervous system development .
Neuronal differentiation during embryonic brain development is governed by a number of mechanisms, one of them is the molecular downstream of Pax6 . Pax6 regulates brain development by preventing depletion of NPCs, and also regulates the process of differentiation into glutamatergic neuron through inducing Tbr2, Ngn2, NeuroD1 and Tbr1 [9,10,11,12]. Pax6 is expressed in NPCs during cortical neurogenesis, making Pax6 a widely used marker for NPCs . Heins et al.  first determined that the overexpression of Pax6 in cortical NPCs deters their proliferation and induces neuronal differentiation. However, detailed analysis of the cell cycle concluded that Pax6 also plays an essential part in the maintenance of proliferative NPCs during corticogenesis . These showed how Pax6 controls the balance between NPC proliferation and differentiation into glutamatergic neuron in the cortex .
Valproic acid (VPA) is observed to influence the proliferation and differentiation of neuronal cells. VPA acts as an inhibitor for class I and II HDACs through activating transcription from diverse promoters and causes hyperacetylation of histones. Interestingly, this inhibition of HDAC curbs proliferation and promotes neuronal differentiation of adult hippocampal neuronal progenitor cells . VPA also directly or indirectly inhibits GSK-3β activity altering the Wnt/β-catenin signaling, and β-catenin, as a result, affects the regulation of the ERK pathway. VPA is generally considered as a neuroprotective drug through its regulation of the mentioned pathways, including LOX, PPARs, and PTEN pathways .
This study is aimed to examine the effect of VPA administration in the expression of TERT during early embryonic development and in cultured NPCs. Moreover, the mechanism regulating TERT expression in NPCs and the alteration of synaptic protein expression by regulating TERT expression are also investigated. We hypothesize that the expression of TERT is upregulated by VPA during development through histone deacetylase inhibition. Transfection of
The materials used in this study's experiments are listed below along with their respective suppliers: Lithium chloride, Sodium butyrate, Trichostatin A, and Valproic acid from Sigma (St. Louis, MO); ECL™ reagents from Amersham Life Science (Arlington Heights, IL); Trizol® reagent, SuperScript™II Reverse Transcriptase,
Antibodies from the following companies: β-actin antibody from Sigma (St. Louis, MO); Tuj-1 antibody from Covance (Princeton, NJ); TERT antibody from Santa Cruz Biotechnology (Santa Cruz, CA); vGluT1, PSD-95, α-CaMKII, NeuroD1, Mash1, BRG1, Synaptophysin antibody from Abcam (Cambridgeshire, England); GAD, Nestin, GFAP, Pax6 and Ngn2GFAP antibody from Millipore (Billerica, MA); HDAC1, Histone H3, Acetyl-Histone H3, GSK3β and phospho-GSK3β antibody from Cell signaling (Boston, MA).
Time-determined pregnant SD (Sprague-Dawley) rats were obtained from DaeHan BioLink (Daejeon, Korea). Animals were kept in a room with a 12 hour: 12 hour circadian light cycle (starting from 06:00) with constant humidity (55±5%) and constant temperature (22±2℃). Animals were treated and maintained in accordance with the Principle of Laboratory Animal Care (NIH publication No. 8023, revised 1978) and were approved by the Animal Care and Use Committee of Konkuk University, Korea (KU12115, 12016). All efforts were undertaken toward minimizing the number of animals used and their suffering.
The sodium valproate was prepared in 0.9% saline solution (100 mg/ml).Timed pregnant rats were subcutaneously injected with 400 mg/kg valproate on gestational day 12. A subcutaneous injection of saline was used as a control.
The cortical progenitor cells from embryos were prepared following by previous reports [19,20] with minor modifications . NPC cultures were prepared from E14 embryos of SD rats.
siRNA transfection was performed using lipofectamine 2000 (Invitrogen, Carlslab, CA). Briefly,
After transfection, NPCs were lysed with SB (2x SDS-PAGE sample buffer) as previously reported . Isolated cortical tissues were homogenized with RIPA buffer, and the extracted lysates were mixed with SB. Protein concentrations were calculated by BCA assay and 50 µg of proteins for each sample was run in the 10% SDS-PAGE. Polyvinyl alcohol (1%, in PBS) was used for blocking the nitrocellulose membranes containing transferred proteins. The blocked membranes were treated with primary antibody and incubated for 16 hour at 4℃. After incubation for primary antibody, a peroxidase-conjugated secondary antibody (Santa Cruz, CA), with the same species as the primary antibody used, was incubated to the membranes for 2 hour at RT (room temperature). Each band of interest was detected using the ECL™ reagent and visualized by Bio-Rad image analyzer (Bio-Rad, Hemel Hampstead, UK). The loading control used was β-actin for all proteins of interest.
From NPCs or isolated cortical embryonic brain, RNA was extracted with Trizol reagent (Invitrogen, Carlslab, CA). An aliquot of 1 µg from the total RNA was transformed into cDNA by reverse transcription reaction. The DNA bands of interest were amplified using PCR and the PCR products for amplified DNA of interest were run in 1.0% agarose gel. After electrophoresis, agarose gel was stained by ethidium bromide, and visualized by image analyzer. All primers were purchased from Invitrogen, and the sequence of primers used in this study are as below:
NPCs were grown on cover slip, and fixed with 4% PFA (paraformaldehyde) at 4℃ for 2 hour. The cells were immersed in 0.3% Triton X-100 for 15 min at RT and blocked with a blocking buffer for 30 min at RT. The cells were incubated for 16 hour at 4℃ while being treated with primary antibodies. Secondary antibodies conjugated fluorescent protein were treated to the cells for 2 hour at RT. After washing, the NPCs onto cover slip was mounted in Vectashield and visualized by confocal microscope (TCS-SP, Leica, Heidelberg, Germany).
Embryonic brains were fixed with 4% PFAand dehydrated with sucrose before sectioning by cryostat (CM 3050, Leica Instruments). The chosen sections were immersed in 0.1% Triton X-100 for 30 min at RT and blocked for 1 hour with 1% BSA in PBS at RT. These sections were incubated for 16 hour at 4℃ after being treated with primary antibodies. After washing, the sections were incubated with secondary antibodies for 2 hour at RT. After washing, the sections were mounted in Vectashieldand visualized by confocal microscope (TCS-SP, Leica, Heidelberg, Germany).
Immunoprecipitation using NPCs was performed by following report . NPCs were lysed with LB buffer for 15 min and the lysates were centrifuged at 1500 g for 5 min. The supernatant was incubated with antibody for 24 hour at 4℃. After incubation, the sample was incubated with Protein G Agarose (Millipore) for 24 hour at 4℃. After incubation, the supernatant was discarded after centrifugation. The agarose beads in the pellet were washed using LB buffer. The pellet was used for Western blot experiments. For negative control experiments, all procedures were identically handled without any antibody.
We performed chromatin immunoprecipitation based on previous report  with minor modifications . The sequence of primers used in this study are as below:
In utero transfection was performed as previously reported . Briefly, E14.5 mice embryos were used for electroporation after anesthetization with isoflurane.
Data were shown as the mean±standard error of mean (S.E.M) and analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Newman-Keuls test as a posthoc test. Two-way ANOVA was performed to identify the effect of valproic acid treatment or
To investigate the biochemical pathway mediating the effects of VPA on TERT expression, we treated several chemicals on cultured NPCs at DIV1 with the following concentrations: VPA (0.5 mM), trichostatin A (TSA, HDAC inhibitor, 0.2 µM), sodium butyrate (SB, HDAC inhibitor, 0.1 mM), while Lithium Chloride (LiCl, GSK-3β inhibitor, 0.2 mM), and TDZD-8 (GSK-3β inhibitor, 5 µM). VPA, TSA and SB independently increased the expression of TERT proteins in cultured NPCs. On the other hand, TDZD decreased TERT expression [F(5,12)=592.4, p<0.0001] and LiCl did not show any effects on TERT protein level (Fig. 3). In addition, only VPA, TSA, and SB upregulated the expression of AcH3 proteins [F(5,12)=31.48, p<0.0001]. Meanwhile, VPA, LiCl, and TDZD increased the phosphorylation of GSK-3β but not TSA or SB [F(5,12)=21.61, p<0.0001]. These results confirm the properties of VPA as HDAC and GSK-3β inhibitor. Moreover, the HDACi property of VPA is directly involved in the increased expressions of TERT and AcH3 proteins.
We examined the binding of proteins related to chromatin opening onto the promoter regions of
We then examined the binding of proteins related to chromatin opening onto the promoter regions of
To investigate whether TERT has a critical role in VPA-mediated alterations of the expression of synaptic proteins, transient
On culture day 1, TERT, Pax6 and nestin expressions were decreased by
During DIV1 (Fig. 5A), expression of nestin was affected by VPA treatment [F(1,8)=33.86, p=0.0004] and
At DIV7, the Ngn2 expression was significantly changed by VPA treatment [F(1,8)=17.04, p=0.0033] and
At DIV12 (Fig. 5B), the level of synaptic proteins expressed by glutamatergic and GABAergic neurons were examined. Expression level of PSD-95 protein was significantly changed by VPA treatment [F(1,8)=14.50, p=0.0052] and
Protein expression level of synaptophysin was significantly changed by VPA treatment [F(1,8)=711.0, p<0.0001] and
In addition, we performed
The use of
The expression of TERT is agreed to be regulated by intricate regulatory pathways mainly at the transcriptional level, with the remodeling of chromatin and nucleosome organization identified as key factors in the physiological translation . Post-transcription histone acetylation/deacetylation is implicated to be crucial in altering chromatin structure and modulating genetic makeup [25,26]. In human normal somatic cells, histone deacetylase (HDAC) is suggested to be recruited by Sp1 and Sp3, and has associated with
Pax6 was upregulated in NPCs after VPA exposure, as previously reported . Interestingly, in the current study, Pax6 expression was attenuated when the VPA-treated cells were transfected with
It is interesting to note how VPA can regulate TERT proteins bound to
Both telomerase and TERT are expressed in neural precursor cells of rats and mice during embryonic development. Telomerase activity levels noticeably decrease between E13 and E18 until they become undetectable . This decline in telomerase activity goes along with the marked decrease of the proliferation of neuroblasts. On the other hand, TERT activity levels belatedly decline at postnatal day 5, concurring with the occurrence of synapse formation and programmed cell death of neurons . This non-parallel decline of activity levels of telomere and TERT suggest that TERT may have a role in cell protection and synapse formation. In our previous study utilizing TERT-tg mice, we elucidated the effects of TERT overexpression in glutamatergic neuronal differentiation, postsynaptic maturation, and autism-relevant behaviors . Here, we supplement the involvement of TERT in neurodevelopment through in vitro studies showing that
This study further expands the relevance of the VPA model of autism as a strong environmental contributor to ASD with a significant clinical relevance [33,34]. We already learned through various validated rodent models that VPA induces altered neuronal apoptosis , increased neuronal cell progenitor pool and macrocephaly phenotype , increased neurite outgrowth in rat primary cortical neurons , altered glutamate regulations in the hippocampus , cerebellar anomalies , histone hyperacetylation and MeCP2 altered expression [39,40], and imbalance between excitation and inhibition neurotransmission [1,41,42], among others. In the current study, we firstly understood that VPA enhances TERT expression. Moreover, we confirmed that Pax6 is directly affected by TERT but could be independently enhanced by VPA, and possibly by other regulators. Moving forward, it is important to unify the already known effects of VPA in the brain and converge these diverse pathways to help explain the mechanisms of ASD. Moreover, we are also testing therapeutic substances that target the affected pathways in the VPA model and how they reverse the abnormalities in the brain and behavior of this model.