Aberrant epigenetic gene regulation in hippocampal neurogenesis of mouse offspring following maternal exposure to 3,3’-iminodipropionitrile

Abstract

Maternal exposure to 3,3′-iminodipropionitrile (IDPN) affects hippocampal neurogenesis in mouse offspring, with biphasic disruption, which facilitates neurogenesis during exposure and reduces the broad range of the granule cell lineage population at the adult stage. The present study investigated the epigenetically hypermethylated and downregulated genes related to the IDPN-induced disrupted neurogenesis. Mated female mice were treated with IDPN at 0 or 1200 ppm in drinking water from gestational day 6 to postnatal day (PND) 21 on weaning. The hippocampal dentate gyrus of male offspring on PND 21 was subjected to methyl-capture sequencing and real-time reverse transcription-PCR analyses, followed by validation analyses on DNA methylation. Three genes, Edc4, Kiss1 and Mrpl38, were identified as those showing promoter-region hypermethylation and transcript downregulation, with Mrpl38 sustaining the changes through PND 77. Immunohistochemically, MRPL38, a mitochondrial ribosomal protein, revealed an irreversible decrease in the number of immunoreactive interneurons in the dentate gyrus hilar region, suggesting a causal relationship with the long-lasting effect on neurogenesis by the impaired migration due to mitochondrial dysfunction of interneurons, which regulate the differentiation and survival of granule cell lineages. Downregulation of Edc4 may also be responsible for decreased neurogenesis on PND 77 owing to a mechanism involving interleukin-6 downregulation via processing body dysfunction. Downregulation of Kiss1 may be responsible for the facilitation of neurogenesis during IDPN-exposure due to decreased glutamatergic neurotransmission and also for suppressed neurogenesis on PND 77 due to decreased expression of immediate-early genes, which play a crucial role in the maintenance of cell differentiation or plasticity.

INTRODUCTION

The adult mammalian brain has the ability to generate new neurons originating from neural stem cells and progenitor cells in the subgranular zone (SGZ) of the hippocampus and the subventricular zone of the lateral ventricles, which is called adult neurogenesis (Kempermann et al., 2004; Ming and Song, 2011). As new granule cell neurons from the SGZ become integrated into the dentate gyrus, hippocampal neurogenesis is an important mechanism in spatial learning and memory in the adult brain (Pan et al., 2013). In the SGZ, type-1 neural stem cells undergo self-renewal to differentiate into type-2a, type-2b and type-3 proliferative progenitor cells (Kempermann et al., 2004). Type-3 progenitor cells differentiate into postmitotic immature granule cells, and then finally into mature granule cells that populate the granule cell layer (GCL; Hodge et al., 2008). It is reported that γ-aminobutyric acid (GABA)-ergic interneurons in the hilus of the dentate gyrus control granule cell differentiation and regulate the maintenance of an appropriate granule cell population (Lussier et al., 2009; Masiulis et al., 2011). In addition to GABAergic interneuron inputs, the dentate gyrus receives various types of projections from other brain regions, such as cholinergic, dopaminergic, noradrenergic, serotonergic and glutamatergic inputs (Freund and Buzsáki, 1996; Masiulis et al., 2011). Both cholinergic and glutamatergic inputs to the SGZ are important for maintaining the appropriate proliferation and differentiation of granule cell lineages (Cameron et al., 1995; Freund and Buzsáki, 1996).

3,3’-Iminodipropionitrile (IDPN) is a neurotoxic compound known to cause proximal axonopathy in the nervous system of rodents. It is reported that IDPN causes proximal axonal swelling followed by distal axonal atrophy, accompanied by an impairment of slow axonal transport resulting in accumulation of neurofilaments (Clark et al., 1980; Griffin et al., 1978). IDPN is also reported to produce an irreversible behavioral syndrome in rodents (Selye, 1957), and the severity of IDPN-induced behavioral deficits and the degeneration of vestibular cells in the crista ampullaris showed a correlation (Khan and Ibrahim, 2015). Developmental exposure to IDPN has also been shown to cause morphometric changes in hippocampal structures (Zmarowski et al., 2012). With regard to the developmental neurotoxicity of IDPN, we previously found that maternal exposure to IDPN reversibly disrupted neurogenesis targeting late-stage differentiation of granule cell lineages in the dentate gyrus in rat offspring (Itahashi et al., 2015). However, we have recently found that maternal IDPN exposure in mice affected hippocampal neurogenesis, increasing the number of postmitotic granule cells involving glutamatergic signals at the end of maternal exposure, and then suppressing SGZ cell proliferation to result in reduction of the broad range of the granule cell lineage population in offspring (Hasegawa-Baba et al., 2017).

Recently, many studies have reported that various epigenetic mechanisms, including DNA methylation, histone modifications and non-coding RNAs are involved in the regulation of adult neurogenesis (Sun et al., 2011). Among these epigenetic mechanisms, DNA methylation is known for its role in long-term gene silencing or downregulation, which serve as the basis of the establishment of cell fate (Klose and Bird, 2006). This relationship is particularly common in CpG sites at promoter regions, where DNA methylation may directly interfere with transcription factor binding to DNA or indirectly suppress transcription through methylated DNA binding proteins that recruit histone deacetylases, leading to chromatin condensation and subsequent gene silencing (Jones et al., 1998). During the neural induction of embryonic stem cells to neural stem cells, pluripotency genes are methylated and silenced, suggesting that DNA methylation plays an important role in neurogenesis (Mohn et al., 2008). Although the results of epigenetic changes in neurogenesis have remained unexplored, environmentally induced disruption of DNA methylation warrants further study (Ceccatelli et al., 2013), given the clear importance of DNA methylation to neuronal development. In fact, exposure to stress (Mueller and Bale, 2008), toxicants (Kundakovic et al., 2013), and maternal neglect (Weaver et al., 2004) in early life have been shown to disrupt epigenetic programming involving DNA methylation in the brain, with lasting consequences for brain gene expression and behavior.

In the present study, we examined the effects of maternal IDPN exposure via an epigenetic mechanism on hippocampal neurogenesis in the dentate gyrus in offspring mice. For this purpose, the hippocampal dentate gyrus of male offspring was first subjected to a search for genes with downregulated transcripts induced by promoter region hypermethylation. We then examined the reversibility of the methylation status, transcript levels, and cellular distribution of the corresponding proteins in the dentate gyrus.

MATERIALS AND METHODS

Chemicals and animals

IDPN (CAS No. 111-94-4, purity > 97%) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Forty-eight pregnant Slc:ICR mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) at gestational day (GD) 1 (the appearance of vaginal plug was designated as GD 0). Animals were individually housed with their offspring in polycarbonate cages with paper bedding until day 21 after delivery [postnatal day (PND) 21 (day of delivery is PND 0)]. Animals were kept in an experimental animal room under the condition of temperature: 23 ± 2°C, relative humidity: 55 ± 15%, 12-hr light/dark cycle. Animals were allowed access to a pelleted basal diet (MF; Oriental Yeast Co., Ltd. Tokyo, Japan) and tap water ad libitum until the start of exposure to IDPN during the experimental period. From PND 21 onwards, offspring were housed with three or four animals per cage and provided with the pellet MF basal diet and tap water ad libitum.

Experimental design

All animal experiments were conducted in accordance with the “Guidelines for Proper Conduct of Animal Experiments” (Science Council of Japan, June 1, 2006), and the protocols were approved by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology.

The present two experiments were identical to those previously reported (Hasegawa-Baba et al., 2017). In experiment 1, pregnant mice were randomly assigned to three groups of 12 animals per group and treated with the water containing 0-, 600-, or 1200-ppm IDPN from GD 6 to PND 21. The high dose of IDPN was determined to induce slight maternal toxicity according to the OECD guideline for the testing of chemicals, based on preliminary study results (Hasegawa-Baba et al., 2017). Body weights and water consumption of dams were measured at 1 or 2 times per week throughout the experimental period. Because neurogenesis is influenced by circulating levels of steroid hormones during the estrous cycle (Pawluski et al., 2009), male offspring were selected for analysis of hippocampal neurogenesis, and at PND 4, the litter size was adjusted by random culling to a uniform litter size of eight pups per litter with as many males as possible. On PND 21, 12 male offspring per group (1 or 2 males per dam) were subjected to perfusion fixation for immunohistochemistry through the left cardiac ventricle with ice-cold 4% (w/v) paraformaldehyde (PFA) in 0.1M phosphate buffer (pH 7.4) at a flow rate of 10 mL/min after deep anesthetization with CO₂/O₂. For DNA and total RNA extraction, 23 male pups each from 0- and 600-ppm groups and 8 male pups from 1200-ppm group (1 to 5 pups per dam) were euthanized by exsanguination from the abdominal aorta under CO₂/O₂ anesthesia and subjected to necropsy. The remaining offspring were maintained without administration of IDPN until PND 77, and body weight was measured once a week. On PND 77, 12 male offspring per group (1 or 2 males per dam) were subjected to perfusion fixation with 4% PFA buffer solution for immunohistochemistry at a flow rate of 10 mL/min in the same manner as PND 21. For DNA and total RNA extraction, brain samples of other male offspring per group (1 to 4 male pups per dam) were prepared.

To obtain additional DNA samples for validation of DNA methylation status of selected genes, an additional IDPN-exposure study (experiment 2) was performed. Pregnant mice were randomly divided into two groups. Six dams per group were treated with 0 (untreated controls) or 1200-ppm IDPN in the drinking water from GD 6 to PND 21. At PND 4, the litter size was adjusted by random culling to a uniform litter size of eight pups per litter as many males as possible. On PND 21, brains of all offspring were collected at autopsy. Seventeen male offspring per group (2 or 3 male pups per group) were prepared. The remaining brains were used for other experimental purposes.

In both experiment 1 and 2, maternal animals were euthanized by exsanguination from the abdominal aorta under CO₂/O₂ anesthesia on the day 21 after delivery.

DNA and RNA Extraction

For DNA and RNA extraction, male offspring brains were removed under CO₂/O₂ anesthesia on PND 21 and PND 77, fixed with methacarn solution for 5 hr at 4°C, and then dehydrated in ice-cold absolute ethanol overnight at 4°C, as described previously (Akane et al., 2013). A coronal brain slice obtained from a position at −2.2 mm from the bregma was prepared. Portions of the hippocampal dentate gyrus were collected using a biopsy punch (Ф1.0 mm; Kai Industries Co. Ltd., Gifu, Japan) and stored in ethanol at −80°C until used for extraction.

For analyses of DNA methylation and transcript expression, genomic DNA and total RNA were extracted from tissue samples of the 0-ppm controls and the 1200-ppm IDPN-exposed group on PND 21 and PND 77 using an All-prep DNA/RNA mini kit (Qiagen, Hilden, Germany). Extracted DNA was used for methyl-capture sequencing (MethylCap-seq) analysis (n = 3 from different dams/group, pooled as one sample), quantitative methylation-specific polymerase chain reaction (PCR) analysis (n = 5 from different dams/group), and pyrosequencing assays (n = 4 from different dams/group). Extracted total RNA was used for real-time reverse transcription (RT)-PCR analysis (n = 6/group).

MethylCap-seq analysis

MethylCap-seq analysis was performed in accordance with the manufacturers’ provided protocols using pooled genomic DNA sample of the 0-ppm controls and the 1200-ppm IDPN-exposed group on PND 21. In brief, genomic DNA was fragmented using a Bioruptor UCD-250 sonicator (Cosmo Bio Co. Ltd., Tokyo, Japan), and methylated DNA was enriched with methyl-CpG binding domain 2 protein (MBD2) using an EpiXplore^TM Methylated DNA Enrichment kit (Clontech Laboratories, Inc., Mountain View, Canada). Subsequently, enriched methylated DNA was used to construct libraries for sequencing using a DNA NEB^® Next ChIP-Seq Library Prep Master Mix Set for Illumina^® (New England Biolabs, Inc., Ipswich, MA, USA). The libraries, one from 0-ppm controls and the other from 1200-ppm IDPN-exposed group, were sequenced using a Miseq Sequencing System (Illumina, Inc., San Diego, CA, USA), and then data analysis was performed using Strand NGS next generation sequencing analysis software (Strand Genomics, Inc., San Francisco, CA, USA). The genomic regions showing hypermethylation of CpG sites in the promoter region up to 2500 bp upstream from the transcription start site of the genes were selected using an enriched region detection algorithm with the criterion that the enrichment factor (ratio of IDPN-exposed sample read counts / control sample read counts) was greater than 3.

Transcript expression analysis of candidate genes

Real-time RT-PCR quantification of mRNA was performed for genes selected as being hypermethylated using the MethylCap-seq data analysis with the RNA samples isolated from the dentate gyrus samples of the 0-ppm controls and the 1200-ppm IDPN-exposed group on PND 21. The genes showing transcript downregulation on PND 21 were also analyzed for expression on PND 77. First-strand complementary DNA was synthesized using SuperScript^® III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) in a total reaction mixture of 20 μL from 1.0 μg of total RNA. Analysis of the transcript levels for candidate genes listed in Supplementary Table 1 was performed using the PCR primers designed with Primer Express software Ver. 3.0 (Thermo Fisher Scientific). Real-time RT-PCR with Power SYBR^® Green PCR Master Mix (Thermo Fisher Scientific) was conducted using a StepOnePlus^TM Real-time PCR System (Thermo Fisher Scientific). The relative differences in gene expression between the 0-ppm controls and the 1200-ppm IDPN-exposed group were calculated using threshold cycle (C_T) values that were first normalized to those of Gapdh or Hprt, which served as endogenous controls in the same sample, and then relative to a control C_T value using the 2^−ΔΔC_T method (Livak and Schmittgen, 2001).

Quantitative methylation-specific PCR

Five genes (Edc4, Kiss1, Mrpl38, Stard3 and Zfp74) were selected for quantitative methylation-specific PCR analysis with the DNA samples isolated from the 0-ppm controls and the 1200-ppm IDPN-exposed group on PND 21. The isolated genomic DNA was sonicated using a Bioruptor UCD-250 sonicator (Cosmo Bio Co., Ltd.), mixed with incubation buffer, and then denatured with heat. Twenty percent of the mixture was stored as input DNA at −20°C until use. The remaining mixture was incubated with MBD2/magnetic bead complexes and then eluted. The methylation-enriched DNA was purified using an EpiXplore Methylated DNA Enrichment kit (Clontech Laboratories, Inc.). Input and methylation-enriched DNA samples were used as templates for quantitative measurement of methylation at target CpG sites by real-time PCR using Power SYBR^® Green PCR Master Mix (Thermo Fisher Scientific) and a StepOnePlus Real-time PCR System (Thermo Fisher Scientific). The PCR primers for the target gene CpG sites were designed using Methyl Primer Express software Ver. 1.0 (Thermo Fisher Scientific) and Primer Express software Ver. 3.0 (Supplementary Table 2; Thermo Fisher Scientific). The quantification was based on the comparative C_T method and involved a comparison of the C_T values of the methylation-enriched DNA to the C_T values of the input DNA.

Pyrosequencing analysis of candidate genes

The percentages of methylated CpG sites in the target sequences of Edc4, Kiss1 and Mrpl38 were measured with bisulfite-converted DNA using the PyroMark Q24 pyrosequencing system (Qiagen) with the DNA samples isolated from the 0-ppm controls and the 1200-ppm IDPN-exposed group on PND 21 and PND 77. The isolated genomic DNA (n = 4/group) was bisulfite converted with an EpiTect^® Plus DNA Bisulfite kit (Qiagen) and then used as a template (10-20 ng) for biotin PCR reactions utilizing a Qiagen PyroMark PCR kit under the following conditions: 95°C for 15 min, (94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec) × 45 cycles, and 72°C for 10 min. The sequencing reactions were performed using PyroMark Gold Q24 reagents (Qiagen). Specific pyrosequencing primers were designed to amplify CpG sites, which are distributed within multiple CpG islands (CpG observed/expected > 0.6) in promoter regions of target gene, using Pyrosequencing Assay Design software Ver. 2.0 (Supplementary Table 3; Qiagen).

Immunohistochemistry in the brain

For immunohistochemical analysis of candidate genes on PND 21 and PND 77, perfusion-fixed brains (n = 8–10/group) were additionally fixed by permeation with 4% PFA overnight. Two mm coronal slices were prepared at −2.2 mm from bregma. Brain slices were further permeation-fixed with 4% PFA overnight at 4°C. Brain slices were processed using a standard protocol for paraffin embedding and were sectioned to a thickness of 3 μm.

Brain sections were subjected to immunohistochemistry using primary antibody against mitochondrial ribosomal protein L38 (MRPL38; rabbit IgG, diluted 1:200; Proteintech Inc., Rosemont, IL, USA). To quench endogenous peroxidase activity, deparaffinized sections were incubated in 0.3% (v/v) hydrogen peroxide solution in absolute methanol for 30 min. The primary antibody was applied overnight at 4°C. Immunodetection was performed using a Vectastain^® Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) with 3,3’-diaminobenzidine/H₂O₂ as the chromogen. Immunostained sections were then counterstained with hematoxylin and coverslipped for microscopic examination. For negative control of immunohistochemistry, the primary antibody was omitted and absence of immunoreactivity was confirmed.

Morphometry of immunolocalized cells

MRPL38⁺ cells distributed within the hilus of the dentate gyrus were bilaterally counted and normalized per area unit of the hilar area (polymorphic layer; Supplementary Fig. 1). Immunolocalized cells were analyzed in an operator-blinded manner. Cornu ammonis 3 (CA3) pyramidal neurons distributed in this area were excluded from quantification. For quantitative measurement of each immunoreactive cellular population, digital photomicrographs at × 100-fold magnification were taken using a BX53 microscope (Olympus Corp., Tokyo, Japan) attached to a DP72 digital camera system (Olympus Corp), and quantitative measurements were performed using WinROOF image analysis and measurement software (version 6.4.2, Mitani Corporation, Fukui, Japan).

Statistical analysis

Numerical data are presented as mean ± SD. Maternal data were analyzed using the individual animal as the experimental unit. Offspring data were analyzed using the litter as the experimental unit. Significant differences between the 0-ppm controls and each IDPN-exposed group were evaluated as follows. Data were analyzed using the Bartlett’s test for homogeneity of variance. If the variance was homogenous, numerical data were assessed using the Dunnett’s test. For heterogeneous data, the Steel’s test was applied. Numerical data consisting of two sample groups were analyzed using the F-test for homogeneity of variance. Student’s t-test was applied when the variance was homogenous between the groups, and the Aspin-Welch’s t-test was performed when data were heterogeneous. Incidence of histopathological changes was compared using the Fisher’s exact probability test. All analyses were performed using the Excel Statistics 2010 software package (Social Survey Research Information Co. Ltd. Tokyo, Japan).

RESULTS

Maternal parameters

Dose-related decreases in the body weight of dams were observed at ≥ 600 ppm during the exposure period (Supplementary Fig. 2). A total of five non-pregnant animals, one from 0-ppm controls and four from 1200-ppm IDPN-exposed group, as confirmed by examination of implantation sites, were excluded from the experiment. At necropsy on day 21 after delivery, body weight was significantly lower at ≥ 600 ppm as compared with the 0-ppm controls. Absolute brain weight was significantly lower at 1200 ppm as compared with the 0-ppm controls (Supplementary Table 4). Histopathologically, axonal degeneration of the spinal cord was observed in the IDPN-exposed dams with statistically significant increase in the incidence at 1200 ppm as compared with the 0-ppm controls (Supplementary Table 4). There were no abnormalities in the gait and behaviors of dams in both IDPN-exposed groups.

Clinical observation, necropsy and histopathological data of male offspring

On PND 21, male offspring at ≥ 600 ppm showed significantly decreased body weight as compared with the 0-ppm controls (Supplementary Table 5). With regard to brain weight in male offspring on PND 21, absolute weight was also significantly lower at 1200 ppm as compared with the 0-ppm controls. On PND 77, body weight and absolute brain weight of male offspring were not significantly different between the 0-ppm controls and each IDPN-exposed group. No abnormalities in the gait and behaviors of offspring were observed in any group before necropsy on PND 21 and after weaning, and there was no notable histopathological change in both IDPN-exposed groups.

Hypermethylated genes detected by MethylCap-seq analysis

Twenty-two CpG sites located at the promoter region up to 2500-bp upstream from the transcription start site of the gene sequence were found to show ≥ 3-fold increase in methylation signals in the 1200-ppm IDPN-exposed offspring compared with the 0-ppm controls on PND 21. Genes downstream of the promoter regions including hypermethylated CpG sites are listed in Table 1.

Table 1. List of hypermethylated genes in the hippocampal dentate gyrus of male offspring on PND 21 after maternal exposure to 1200-ppm IDPN as determined by MethylCap-seq analysis (≥ 3-fold).

Genome location	Accession No.	Gene symbol	Description	Position to TSS
Chr8: 108402762	MGI: 2446249	Edc4	Enhancer of mRNA decapping 4	2130
Chr6: 28380259	MGI: 1921625	Gcc1	Golgi coiled coil 1	1955
Chr7: 140899054	MGI: 1913415	Bccip	BRCA2 and CDKN1A interacting protein	1870
Chr11: 116001853	MGI: 1926269	Mrpl38	Mitochondrial ribosomal protein L38	1781
Chr10: 119640807	MGI: 1921164	Irak3	Interleukin-1 receptor-associated kinase 3	1705
Chr13: 97242609	MGI: 2444730	Ankdd1b	Ankyrin repeat and death domain containing 1B	1614
Chr2: 4574383	MGI: 1914479	Prpf18	Pre-mRNA processing factor 18	1466
Chr5: 130843968	MGI: 2155987	Caln1	Calneuron 1	1219
Chr4: 140975403	MGI: 1352494	Hspb7	Heat shock protein family, member 7 (cardiovascular)	1210
Chr11: 116002226	MGI: 1922033	Fbf1	Fas (TNFRSF6) binding factor 1	1154
Chr1: 135217320	MGI: 2663985	Kiss1	KiSS-1 metastasis-suppressor	1056
Chr4: 9770363	MGI: 95689	Gdf6	Growth differentiation factor 6	993
Chr3: 113062619	MGI: 88020	Amy2a5	Amylase 2a5	864
Chr7: 110906670	MGI: 3030464	Olfr630	Olfactory receptor 630	806
Chr5: 105412411	MGI: 1351624	Abcg3	ATP binding cassette subfamily G member 3	780
Chr7: 6683652	MGI: 104748	Peg3	Paternally expressed 3	520
Chr6: 89971214	MGI: 2148509	Vmn1r47	Vomeronasal 1 receptor 47	513
Chr13: 9875766	MGI: 88398	Chrm3	Cholinergic receptor, muscarinic 3, cardiac	464
Chr19: 29395253	MGI: 1351595	Insl6	Insulin-like 6	406
Chr10: 86956619	MGI: 96919	Ascl1	Achaete-scute family bHLH transcription factor 1	295
Chr7: 30715456	MGI: 107784	Zfp74	Zinc finger protein 74	206
Chr11: 98242528	MGI: 1929618	Stard3	START domain containing 3	102

Abbreviations: Chr, chromosome; IDPN, 3,3′-iminodipropionitrile; PND, postnatal day; TSS, transcriptional start site.

Transcript expression changes in the dentate gyrus

Among the 22 genes selected as showing ≥ 3-fold increases in methylation signals compared with the 0-ppm controls on PND 21, the transcript levels of Edc4, Kiss1, Mrpl38 and Stard3 were decreased in 1200-ppm IDPN-exposed offspring on PND 21 compared with the 0-ppm controls after normalization with either Gapdh or Hprt. While statistically significant difference was not attained, the transcript level of Zfp74 showed a tendency to decrease in 1200-ppm IDPN-exposed offspring compared with the 0-ppm controls after normalization with Hprt. On PND 77, the transcript level of Mrpl38 was also decreased in 1200-ppm IDPN-exposed offspring compared with the 0-ppm controls after normalization with both of Gapdh and Hprt (Table 2).

Table 2. Transcript levels of candidate genes and related genes in the hippocampal dentate gyrus of male offspring on PND 21 and PND 77 after maternal exposure to 1200-ppm IDPN.

		IDPN in drinking water (ppm)
		0							1200
		Relative transcript level normalized to							Relative transcript level normalized to
		Gapdh			Hprt				Gapdh			Hprt
PND 21
No. of animals examined		6			6				6			6
	Abcg3	1.10	±	0.59	1.06	±	0.41		2.11	±	1.60	1.66	±	0.93
	Amy2a5	1.07	±	0.34	1.03	±	0.81		1.11	±	0.75	0.81	±	0.35
	Ankdd1b	1.16	±	0.58	1.17	±	0.66		6.97	±	7.80	6.05	±	7.04
	Ascl1	1.02	±	0.21	1.05	±	0.33		1.03	±	0.39	0.80	±	0.25
	Bccip	1.03	±	0.30	1.01	±	0.15		1.03	±	0.16	0.80	±	0.07
	Caln1	1.01	±	0.14	1.03	±	0.26		1.05	±	0.47	0.83	±	0.37
	Chrm3	1.01	±	0.18	1.02	±	0.21		1.09	±	0.38	0.85	±	0.28
	Edc4	1.01	±	0.16	1.01	±	0.15		0.93	±	0.12	0.74	±	0.08**
	Fbf1	1.00	±	0.09	1.01	±	0.16		1.04	±	0.26	0.80	±	0.07
	Gcc1	1.01	±	0.12	1.01	±	0.16		1.36	±	0.36	1.05	±	0.18
	Gdf6	1.12	±	0.68	1.13	±	0.75		4.65	±	4.90	3.37	±	3.23
	Hspb7	1.09	±	0.59	1.13	±	0.68		3.73	±	3.51	2.78	±	2.51
	Il6	1.08	±	0.44	1.07	±	0.41		17.66	±	27.10	11.86	±	13.15
	Insl6	1.06	±	0.39	1.04	±	0.29		6.95	±	7.27	5.11	±	4.92
	Irak3	1.06	±	0.47	1.08	±	0.53		2.81	±	2.32	1.89	±	1.51
	Kiss1	1.02	±	0.22	1.02	±	0.24		0.68	±	0.68*	0.54	±	0.59*
	Kiss1r	1.02	±	0.21	1.00	±	0.10		0.88	±	0.27	0.71	±	0.29
	Mrpl38	1.01	±	0.16	1.01	±	0.11		0.99	±	0.19	0.78	±	0.18*
	Olfr630	1.02	±	0.25	1.01	±	0.14		3.69	±	3.87	3.30	±	3.61
	Peg3	1.02	±	0.21	1.00	±	0.10		1.23	±	0.13	0.97	±	0.15
	Prpf18	1.01	±	0.20	1.00	±	0.08		1.24	±	0.41	0.94	±	0.14
	Stard3	1.01	±	0.14	1.00	±	0.10		0.88	±	0.25	0.70	±	0.24*
	Vmn1r47	1.01	±	0.15	1.01	±	0.17		4.36	±	4.81	3.87	±	4.33
	Zfp74	1.01	±	0.17	1.02	±	0.25		0.99	±	0.15	0.78	±	0.12
PND 77
No. of animals examined		6			6				6			6
	Edc4	1.02	±	0.25	1.02	±	0.23		1.02	±	0.30	0.87	±	0.14
	Il6	1.15	±	0.64	1.37	±	0.25		0.22	±	0.16	0.16	±	0.11
	Kiss1	1.01	±	0.15	1.05	±	0.39		0.76	±	0.24	0.75	±	0.34
	Kiss1r	1.02	±	0.26	1.04	±	0.32		1.00	±	0.16	0.96	±	0.17
	Mrpl38	1.00	±	0.11	1.03	±	0.31		0.81	±	0.12*	0.59	±	0.07*

Abbreviations: Abcg3, ATP binding cassette subfamily G member 3; Amy2a5, amylase 2a5; Ankdd1b, ankyrin repeat and death domain containing 1B; Ascl1, achaete-scute family bHLH transcription factor 1; Bccip, BRCA2 and CDKN1A interacting protein; Caln1, calneuron 1; Chrm3, cholinergic receptor, muscarinic 3, cardiac; Edc4, enhancer of mRNA decapping 4; Fbf1, Fas (TNFRSF6) binding factor 1; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Gcc1, golgi coiled coil 1; Gdf6, growth differentiation factor 6; Hprt, hypoxanthine guanine phosphoribosyl transferase; Hspb7, heat shock protein family, member 7 (cardiovascular); IDPN, 3,3′-iminodipropionitrile; Il6, interleukin 6; Insl6, insulin-like 6; Irak3, interleukin-1 receptor-associated kinase 3; Kiss1, KiSS-1 metastasis-suppressor; Kiss1r, KISS1 receptor; Mrpl38, mitochondrial ribosomal protein L38; Olfr630, olfactory receptor 630; Peg3, paternally expressed 3; PND, postnatal day; Prpf18, pre-mRNA processing factor 18; Stard3, START domain containing 3; Vmn1r47, vomeronasal 1 receptor 47; Zfp74, zinc finger protein 74.^a Mean ± SD. *P < 0.05, ** P < 0.01, significantly different from 0-ppm controls by Student’s or Aspin-Welch’s t-test.

Validation of hypermethylation status by quantitative methylation-specific PCR

The methylation status of Kiss1 and Mrpl38 significantly increased in the 1200-ppm IDPN-exposed offspring compared with 0-ppm controls on PND 21, whereas Edc4, Stard3 and Zfp74 displayed no change in methylation status on this time point (Fig. 1).

Fig. 1

Quantitative methylation-specific PCR data of selected genes in the hippocampal dentate gyrus of male offspring on postnatal day (PND 21) after maternal exposure to 1200-ppm 3,3′-iminodipropionitrile (IDPN). Values are expressed as the mean + SD. N = 5/group. *P < 0.05, ** P < 0.01, significantly different from 0-ppm controls by Student’s or Aspin–Welch’s t-test.

Evaluation of DNA methylation status by pyrosequencing

In Edc4, Kiss1 and Mrpl38, pyrosequencing of nucleotides 272–301, 151–190 and 187–233 from the transcription start site, respectively, revealed CpG sites numbered as 1–6 in Edc4, 1–3 in Kiss1 and 1–6 in Mrpl38 (Fig. 2).

Fig. 2

Pyrosequencing results for Edc4, Kiss1 and Mrpl38 in the hippocampal dentate gyrus of male offspring on PND 21 after maternal exposure to 1200-ppm IDPN. All cytosine bases within CpG sites were numbered as #1–3 or #1–6. White columns, 0-ppm controls; black columns, 1200-ppm 3,3′-iminodipropionitrile (IDPN). Values are expressed as the mean ± SD. N = 4/group. *P < 0.05, ** P < 0.01, significantly different from 0-ppm controls by Student’s or Aspin–Welch’s t-test.

On PND 21, cytosine bases in site numbers 1 and 2 of Edc4, 2 of Kiss1, and 1 and 4 of Mrpl38 carried greater levels of methylation in the 1200-ppm IDPN-exposed group than in the 0-ppm controls.

On PND 77, cytosine bases in site number 4 of Mrpl38 carried greater levels of methylation in the 1200-ppm IDPN-exposed group than in the 0-ppm group. Methylation levels of cytosine bases in Edc4 and Kiss1 were unchanged between the two groups.

Distribution of immunolocalized cells in the hippocampus

In the control offspring, MRPL38 immunoreactivity was observed within the cytoplasm of neurons distributed in the hilus or GCL in the dentate gyrus on PND 21 and PND 77 (Fig. 3). IDPN exposure at 1200 ppm significantly decreased the density of MRPL38⁺ cells in the hilus on both PND 21 and PND 77 compared with the 0-ppm controls.

Fig. 3

Distribution and number of immunoreactive cells for mitochondrial ribosomal protein L38 (MRPL38) in the hilus of the hippocampal dentate gyrus of male offspring on PND 21 and PND 77 after maternal exposure to 1200-ppm IDPN. Representative images from 0-ppm controls and the 1200-ppm group are shown. Magnification: 200 ×; bar = 100 μm. Graphs show the number of immunoreactive cells/unit area (mm²) of the hilus of bilateral hemispheres. Values are expressed as the mean + SD. N = 8–10/group. *P < 0.05, **P < 0.01, significantly different from 0-ppm controls by Student’s or Aspin–Welch’s t-test.

DISCUSSION

As aforementioned, maternal IDPN exposure at 1200 ppm from GD 6 to PND 21 increased postmitotic neuron-specific NeuN⁺ granule cells in the SGZ and GCL of mouse offspring on weaning (Hasegawa-Baba et al., 2017). Interestingly, the IDPN-exposed offspring at the adult stage showed suppressed SGZ cell proliferation and a reduction in the broad range of the granule cell lineage population, indicating a late effect on hippocampal neurogenesis (Hasegawa-Baba et al., 2017). It is reported that early stress during the lactating period induces a late effect on hippocampal neurogenesis by altered epigenetic regulation in the expression of brain-derived neurotrophic factor in the hippocampus (Suri et al., 2013). We hypothesized that maternal IDPN exposure alters epigenetic gene regulation, which results in the late effect on hippocampal neurogenesis. In the present study, using MethylCap-seq analysis we found that 22 genes showed promoter-region hypermethylation at CpG sites in the offspring hippocampal dentate gyrus following maternal IDPN exposure at 1200 ppm. Among these candidate genes, we further confirmed transcript downregulation of Edc4, Kiss1, Mrpl38 and Stard3 on PND 21 and Mrpl38 on PND 77. Of note, the present IDPN-exposed offspring at 1200 ppm showed body and brain growth suppression. It is reported that undernutrition is considered to suppress methylation status through decreased methyl donors from diet and DNA methyltransferase activity (Zhang, 2015). Therefore, induction of hypermethylation in specific genes even under the growth-suppressed condition due to systemic chemical toxicity at high doses may support the causal relationship to neurotoxic effect rather than undernutrition-related effect. In the present study, validation analysis in methylation-specific quantitative PCR and/or pyrosequencing confirmed the promoter-region hypermethylation of Edc4, Kiss1 and Mrpl38 on PND 21 and sustained hypermethylation of Mrpl38. Obtained results suggest that hypermethylation-related transcript downregulation was sustained with Mrpl38 through the adult stage, while it was transiently observed with Edc4 and Kiss1 at the end of maternal IDPN-exposure.

With regard to Mrpl38 encoding mitochondrial ribosomal protein L38 (MRPL38), we found a decreased number of hilar interneurons expressing MRPL38 at both PND 21 and PND 77 after maternal IDPN exposure by immunohistochemical analysis. This protein is an important constituent of the large subunit of mitochondrial ribosomes responsible for assembling mitochondrial DNA-coded proteins essential for oxidative phosphorylation (Brown et al., 2014). The L38 protein maintains the core architecture of the large subunit’s central protuberance, which interacts with the small subunit and with mitochondrial transfer RNAs bound to the ribosome, and is hence critical for mitochondrial translation (Brown et al., 2014). While the exact function of MRPL38 is still not clear, a recent exome analysis study of human cases revealed a strong association of low-frequency MRPL38 variants with white matter hyperintensities (WMH) on brain magnetic resonance imaging (Jian et al., 2018). These abnormalities are believed to reflect demyelination and axonal loss as a result of chronic ischemia and blood-brain barrier dysfunction (Pantoni and Simoni, 2003). Interestingly, the structure of the L38 protein is similar to the phosphatidylethanolamine-binding proteins (PEBPs) that have been identified in numerous tissues and which have various functions, including roles in neural development and differentiation, which have been implicated in Alzheimer’s disease and gliomas (Ling et al., 2014). Evidence is also accumulating that mutation of mitochondrial ribosomal protein genes is involved in impaired mitochondrial translation leading to neurological diseases (Boczonadi and Horvath, 2014). Of note, MRPL18, another mitochondrial ribosomal protein gene that was found to be upregulated in active multiple sclerosis lesions (Fischer et al., 2012), was also differentially expressed in brains of young spontaneously hypertensive stroke–prone rats and associated with WMH (Lopez et al., 2015). Taken together, these data provide biological plausibility supporting the role of MRPL38 dysfunction in the pathophysiologic changes in the nervous system induced by maternal IDPN exposure.

Chronic IDPN intoxication results in the swelling of proximal axons associated with a disorientation of neurofilaments and central clustered microtubules and mitochondria (Clark et al., 1980). These changes were considered to be due to the impairment of slow axonal transport, which also carries mitochondria (Clark et al., 1980). However, degeneration of mitochondria was not observed under electron microscopic examination (Clark et al., 1980), except for one study reporting the appearance of rounded osmiophilic bodies, 0.1–0.8 microns in diameter, in the mitochondria of both neurons and glial cells in IDPN-treated cats (Fiori and Lowndes, 1988). Recently, many studies have shown that mitochondria play a crucial role in multiple aspects of neuronal development including differentiation and synaptogenesis, which are also important processes in adult neurogenesis (Bertholet et al., 2013; Wang et al., 2014). Considering that interneurons require appropriate mitochondrial function for neuronal migration (Lin-Hendel et al., 2016), decreased MRPL38 expression in interneurons may cause long-lasting effects on neurogenesis through the impairment of appropriate migration due to mitochondrial dysfunction of interneurons, which regulate the differentiation and survival of granule cell lineages (Lussier et al., 2009; Masiulis et al., 2011).

With regard to Edc4 (enhancer of mRNA decapping 4), corresponding protein EDC4 is the scaffold protein, which is known to play a crucial role in the assembly of mRNA processing bodies (P-bodies; Yu et al., 2005). It is also reported that EDC4 interacts with mRNA-decapping enzyme 1a to mediate degradation of mRNA (Jinek et al., 2008), and with CoA synthase to catalyze the CoA biosynthesis in various cells (Gudkova et al., 2012). Of note, Edc4 knockdown severely reduced the production of interleukin-6 (IL-6) through suppression of EDC4 assembly into P-bodies in human monocytic leukemia cells in vitro (Seto et al., 2015). IL-6 is a proinflammatory cytokine known to modulate neurogenesis, and IL-6 knockdown mice have been shown to reduce cell proliferation and lower progenitor cell survival in the hippocampal dentate gyrus (Bowen et al., 2011). In the present study, values in the transcript level of Il6 were highly variable among individual cases in the 1200-ppm IDPN group on PND 21, 3 of 6 cases showed very high expression values and the other 3 cases showed similar levels to 0-ppm controls. While the cases showing very high Il6 expression suggested induction of neuroinflammation during IDPN-exposure, other cases examined suggested suppression of Il6 upregulation related to Edc4 downregulation through promoter-region hypermethylation. We observed an increase of NeuN⁺ postmitotic granule cells by 1200-ppm IDPN-exposure at this time point (Hasegawa-Baba et al., 2017), and this increase may be the reflection of promoted neurogenesis due to high levels of IL-6 during IDPN-exposure. On PND 77, the Il6 transcript level was decreased in this group compared with 0-ppm controls, while a statistically significant difference was not attained. Il6 downregulation at this time point was probably due to the outcome of the transient Edc4 downregulation observed on PND 21. The Il6 downregulation on PND 77 may be responsible for the decreases in the broad range of the granule cell lineage population due to the suppressed cell proliferation reported in our previous study (Hasegawa-Baba et al., 2017). Although promoter-region hypermethylation and transcript downregulation of Edc4 were only observed on PND 21, the transient Edc4 downregulation may be responsible for decreased neurogenesis in offspring at the adult stage after exposure to IDPN through the suppression of IL-6.

Kisspeptin (KiSS-1 metastasis-suppressor), which is encoded by Kiss1, is known to activate kisspeptin receptors (KISS1R) on gonadotropin-releasing hormone neurons to control puberty and subsequent fertility in the mammal brain (Kirilov et al., 2013). In the hippocampus, one of the KISS1R, GPR54, is highly and exclusively expressed in granule cells (Herbison et al., 2010). However, the functional role of kisspeptin signaling in the hippocampal neurogenesis is unknown. In the present study, we found that maternal exposure to IDPN increased the methylation status of the promoter region and decreased the transcript level of Kiss1 without transcript expression alteration of Kiss1r on PND 21. We further tried to demonstrate kisspeptin-expressing neurons by in situ hybridization and also by immunohistochemistry in the hippocampus in the present study. However, we could not detect any expression signals (data not shown), possibly due to the low level of expression in the hippocampus. Other reports have also shown that immunohistochemical studies failed to detect kisspeptin-immunoreactive neurons or fibers within the hippocampus of rats or mice (Clarkson et al., 2009; Desroziers et al., 2010; Mikkelsen and Simonneaux, 2009). It is noteworthy that Kiss1 mRNA has been shown to be detected in the hippocampus, although its expression level is 50-fold lower than that in the hypothalamus (Muir et al., 2001). Therefore, kisspeptin may exert its function at low levels within the hippocampus. Our pyrosequencing data in the present study revealed that CpG sites of the Kiss1 promoter region were highly methylated, suggesting that the Kiss1 transcript level was epigenetically suppressed in the hippocampus.

Of note, kisspeptin has been demonstrated to activate immediate early genes (IEGs), early growth response factor 1 and FBJ osteosarcoma oncogene (FOS) to regulate gonadotropin genes through Kiss1 receptors (Witham et al., 2013). IEG is shown to mediate transcriptional regulation of neuronal plasticity, including both axonal and synaptic plasticity (Guzowski, 2002). Additionally, kisspeptin interacts with many different neurotransmitter systems in the hippocampus, such as adrenergic, serotoninergic, acetylcholinergic, dopaminergic and GABAergic signalings (Telegdy and Adamik, 2013). These findings agree with our previous results that maternal IDPN exposure at 1200 ppm decreased glutamatergic input to the dentate gyrus at the end of exposure and decreased the number of immunoreactive cells for IEG products, activity-regulated cytoskeleton-associated protein (ARC) and FOS, in the GCL at the adult stage (Hasegawa-Baba et al., 2017). With regard to the decrease in glutamatergic input to the dentate gyrus at the end of IDPN-exposure, we previously discussed this in relation to the proliferation and differentiation of neural stem cells, which result in increased neurogenesis as indicated by the increase in NeuN⁺ postmitotic granule cells at that time point. With regard to the decreased number of ARC⁺ and FOS⁺ granule cells at the adult stage, we previously discussed this in relation to the decreased axonal and synaptic plasticity in the granule cells of adult offspring, likely due to the suppressed production of new neurons, as evidenced by a reduction in the broad range of the granule cell lineage population. Interestingly, late onset of schizophrenia-like behavior was observed in immune-deficient mice accompanied with impairment of both hippocampal neurogenesis and Kiss1 expression (Cardon et al., 2010). From these findings, Kiss1 downregulation at the end of IDPN-exposure may facilitate neurogenesis due to the decrease in glutamatergic neurotransmission during IDPN-exposure and then suppress the neurogenesis as a late effect due to the decrease in the expression of IEGs, which play a crucial role in the maintenance of cell differentiation or plasticity.

In conclusion, global methylation analysis revealed that maternal IDPN exposure at 1200 ppm irreversibly decreased the expression of MRPL38 in interneurons due to transcript downregulation related to promoter-region hypermethylation in mice. Downregulation of this mitochondrial ribosomal protein may cause a long-lasting effect on neurogenesis by impairment of the appropriate migration of interneurons due to mitochondrial dysfunction. Maternal IDPN exposure also caused a transient transcript downregulation of Edc4 and Kiss1 related to promoter-region hypermethylation. Edc4 downregulation may be responsible for decreased neurogenesis at the adult stage owing to the mechanism involving IL-6 downregulation via P-body dysfunction. Kiss1 downregulation may be responsible for the facilitation of neurogenesis due to the suppression of glutamatergic neurotransmission during IDPN-exposure and also for suppressed neurogenesis at the adult stage due to decreased expression of immediate-early genes, which play a crucial role in the maintenance of cell differentiation or plasticity. These alterations of epigenetic regulation may be responsible for long-lasting disruption of neurogenesis in mice maternally exposed to IDPN.

ACKNOWLEDGMENTS

The authors thank Mrs. Shigeko Suzuki for her technical assistance in preparing the histological specimens. This work was supported by Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS; grant No. 25292170).

Conflict of interest

The authors declare that there is no conflict of interest.

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