Transcriptome Analysis of PC12 Cells Reveals That trans-Banglene Upregulates RT1-CE1 and Downregulates abca1 in the Neurotrophic Pathway

Abstract

trans-Banglene and cis(c)-banglene possess neurotrophin-like activity in rat neurons. However, the molecular mechanisms underlying t-banglene-induced neurotrophic activity in rat and human neurons remain unclear. Here, we performed transcriptome analysis in PC12 cells, a rat adrenal gland pheochromocytoma cell line treated with t-banglene, using comprehensive RNA sequencing. The differentially expressed gene analysis of the sequencing data revealed that the expression of RT1 class I, locus CE1 (RT1-CE1) was upregulated, and that of ATP binding cassette subfamily A member 1 (abca1), myosin light chain 6, and hippocampus abundant transcript 1 was downregulated in t-banglene-treated PC12 cells, with statistically significant differences. We also confirmed the RT1-CE1 upregulation and abca1 downregulation in t-banglene-treated PC12 cells by real-time reverse transcription quantitative PCR. RT1-CEl is a major histocompatibility complex class I (MHCI) protein. ABCAl is a major cholesterol transporter that regulates efflux of intracellular cholesterol and phospholipids. Thus, our results suggest an exciting link between MHCI, cholesterol regulation, and neural development.

INTRODUCTION

The chemical components of bangle, Zingiber purpureum (Z. purpureum), rhizome are similar to those of Z. cassumunar, and their compounds possess neurotrophin-like activities. Two phenylbutenoid dimers, trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3′,4′-dimethoxystyryl] cyclohex-1-ene (t-banglene) and cis-3-(3,4-dimethoxyphenyl)-4-[(E)-3′,4′-dimethoxystyryl] cyclohex-1-ene (c-banglene), have been isolated as main components from bangle and shown to possess neurotrophic activities, including neurite outgrowth, neuronal cell growth, and neural viability promoting effects in PC12 cells, a rat adrenal gland pheochromocytoma cell line, and primary cultured rat cortical neurons.^1,2) Additionally, these compounds showed hippocampal neurogenesis in olfactory bulbectomy mice,¹⁾ improved spatial learning, reduced memory deficits, and promoted neurogenesis in the dentate gyrus of senescence-accelerated mouse P8, a mouse model of amyloid precursor protein spontaneous overproduction and oxidative damage.³⁾ These reports suggest that bangle extract containing t-banglene and c-banglene is useful as a therapeutic medicine for the prevention of age-associated diseases and cognitive impairments such as Alzheimer’s disease. Hirano et al. recently reported that bangle extract, t-banglene, and c-banglene enhance neurite outgrowth of human fetal neural stem cells (hfNSCs), and that they upregulate mRNA expression of Wnt signaling-related genes, N-MYC, AXIN2, and EGFR, in hfNSCs,⁴⁾ suggesting that bangle extract and c-banglene induce neurotrophic effects via activation of the WNT/β-catenin signaling pathway in human neurons. However, the molecular mechanisms underlying t-banglene-induced neurotrophic activity in rat and human neurons remain unclear. In this study, we investigated the molecular mechanisms underlying the neurotrophic activity of t-banglene in PC12 cells.

MATERIALS AND METHODS

Preparation of trans-Banglene

Compound 1-(3,4-dimethoxyphenyl) buta-1,3-diene was heated in the presence of hydroquinone in toluene and separated by silica gel column chromatography to yield t-banglene (Fig. 1A), according to the modified method of Tuntiwachwuttikul et al.^1,5) Synthetic t-banglene was dissolved in dimethyl sulfoxide (DMSO).

Fig. 1. Experimental Procedure for Induction of Neurite Outgrowth in PC12 Cells

(A) Chemical structure of trans-3-(3′,4′-dimethoxyphenyl)-4-((E)-3″,4″-dimethoxystyryl) cyclohex-1-ene (t-banglene). (B) Experimental procedure for neurite outgrowth induction in PC12 cells. Cells were seeded on a type-I collagen coated 24-well plate in the growth medium. After one day, neurite outgrowth in PC12 cells was induced in a differentiation medium supplemented with one of the following: 0.3% DMSO, 50 ng/mL native mouse nerve growth factor 2.5S protein (mNGF), or 0.3, 3, or 30 µM t-banglene for nine days of incubation. DMSO or mNGF were used as negative or positive controls, respectively. Each differentiated medium in the wells was changed every three days.

Culture of PC12 Cells

PC12 cells were cultured in growth medium [Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 with L-glutamine, sodium pyruvate, and N′-2 Hydroxyet hylpiperazine-N′-2 ethanesulphonic acid (HEPES) (Nacalai Tesque, Kyoto, Japan) supplemented with 10% horse serum (HS; Sigma-Aldrich, MO, U.S.A.), 5% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, U.S.A.), and 100 units/mL penicillin and 100 µg/mL streptomycin (P/S; Thermo Fisher Scientific)] on a type-I collagen-coated cell culture dish (Nippi, Tokyo, Japan) at 37 °C in the presence of 5% CO₂.

Induction of Neurite Outgrowth in PC12 Cells

Neurite outgrowth in PC12 cells was induced by the addition of t-banglene to a type I collagen-coated plate for nine days. The experimental procedure is illustrated in Fig. 1B. PC12 cells cultured in growth medium on a type-I collagen-coated dish were dissociated into single cells using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Thermo Fisher Scientific). Cells were seeded on a type-I collagen-coated 24-well plate (Nippi) at a density of 1.2 × 10⁴ cells/well in the growth medium. After one day, neurite outgrowth in PC12 cells was induced in the differentiation medium (DMEM/Ham’s F-12 with L-glutamine, sodium pyruvate, and HEPES (Nacalai Tesque) supplemented with 2% HS (Sigma-Aldrich), 1% FBS (Thermo Fisher Scientific), and P/S (Thermo Fisher Scientific)) supplemented with one of the following: 0.3% DMSO, 50 ng/mL native mouse nerve growth factor 2.5S protein (mNGF; Alomone Labs, Jerusalem, Israel), which induces neurite outgrowth of PC12 cells,⁶⁾ or 0.3, 3, or 30 µM t-banglene; the cells were incubated for nine days at 37 °C in the presence of 5% CO₂. DMSO and mNGF were used as the negative and positive controls, respectively. Each differentiated medium in the wells was changed every three days.

Imaging Analysis of Neurite Outgrowth in PC12 Cells

After culturing the induced cells for nine days, as shown in Fig. 1B, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 4 °C. Each well was washed with PBS and photographed under a phase contrast using a fluorescence microscope (BIOREVO BZ-9000, Keyence, Osaka, Japan). Neurite-bearing PC12 cells (Figs. 2A–C, black arrows) were counted, and the percentages of PC12 cells with neurites were calculated based on all cells in the images (n = 5 each).

Fig. 2. Induction of Neurite Outgrowth in PC12 Cells by t-Banglene

After nine days of neuronal differentiation culture, as shown in Fig. 1B, the cells treated with 0.3% DMSO (A), 50 ng/mL mNGF (B), or 0.3 (C, left panel), 3 (C, center panel), or 30 (C, right panel) µM t-banglene, fixed, and photographed under phase contrast using a fluorescence microscope. Black arrows in each image indicate the neurite outgrowth of PC12 cells. The black scale bar in each image represents 50 µm. Images (A–C) are representative of one of three independent experiments. These results were reproducible across the three independent experiments. (D) Neurite-bearing PC12 cells were counted in the images and the percentages of PC12 cells with neurites were calculated based on image total cell count (n = 5 each). Data were represented as means ± S.E.M. of three independent experiments. *** p < 0.001, versus DMSO-treated cells.

Transcriptome Analysis Using a Comprehensive RNA Sequencing

We performed a comprehensive RNA sequencing for transcriptome analysis of t-banglene-treated PC12 cells using a previously reported method.^7–10) Briefly, total RNA was extracted from PC12 cells treated with 0.3% DMSO or 3 µM t-banglene (n = 3 each) and incubated for nine days (Fig. 1B) using a RNeasy Mini Kit (Qiagen, GmbH, Germany). The mRNA-sequencing libraries were constructed from each total RNA extract using a TruSeq Stranded mRNA LT Sample Prep kit (Illumina, CA, U.S.A.) according to the manufacturer’s instructions. Single-end reads of 101 base pairs were generated using a NovaSeq 6000 sequencer (Illumina). RNA sequencing tags that were mapped to Rattus norvegicus reference genome using HISAT2 software were analyzed. Known genes and transcripts were assembled using the StringTie software^8–10) based on a reference genome model. After assembly, the abundance of gene/transcript was calculated in the read count and normalized as fragments per kilobase of transcript per million mapped reads (FPKM) value per sample. For the analysis of differentially expressed genes, read count values of known genes obtained through the −e option of StringTie software were used as the original raw data (Supplementary Table S1). Low-quality transcripts were filtered during data pre-processing. Afterward, log2 transformation of read count + 1 and quantile normalization were performed (Supplementary Table S2). Statistical analysis was performed using the fold change and local pooled error (LPE) test to compare 0.3% DMSO- and 3 µM t-banglene-treated PC12 cells (n = 3 each) (Supplementary Table S2). Significant differentially expressed genes were selected on conditions of |fc| ≥ 2 and LPE test raw p-value < 0.05 (Table 1). Expression levels of these genes in between 0.3% DMSO- and 3 µM t-banglene-treated PC12 cells (Supplementary Table S2) are shown as scatter plots, volume plots, and volcano plot. The normalized expression levels in Table 1 were clustered by similarity of genes and samples (hierarchical clustering analysis) using the Euclidean method and complete linkage, shown as a heatmap.

Table 1. Four Genes Selected by the Differentially Expressed Gene Analysis of Comprehensive RNA Sequencing in DMSO- and t-Banglene-Treated PC12 Cells

Gene	Log2 transformation of read count + 1 and quantile normalization
	0.3% DMSO			3 µM t-Banglene
RT1-CE1	1.001864	1.098910	1.440480	1.004672	4.083357	3.841744
Abca1	2.995232	2.748710	3.054257	1.762184	1.819542	1.674975
Myl6l	3.326853	3.122448	3.846776	0.511211	2.660716	2.661284
Hippocampus abundant transcript 1	5.361765	0.664102	5.301268	0.615717	5.313539	0.570220
Gene	Means (averages)		Standard deviation (S.D.)		DMSO/t-Banglene
	0.3% DMSO	3 µM t-Banglene	0.3% DMSO	3 µM t-Banglene	Fold change	p-Value
RT1-CE1	1.180418	2.976591	0.230388	1.712000	−3.472977	2.82242E-33
Abca1	2.932733	1.752234	0.162078	0.072795	2.266552	1.11273E-05
Myl6l	3.432026	1.944403	0.373442	1.241181	2.804264	0.005997311
Hippocampus abundant transcript 1	3.775711	2.166492	2.694903	2.725517	3.050868	0

Real-Time Reverse Transcription Quantitative PCR (RT-qPCR)

For RT-qPCR, cDNA was synthesized from the total RNA of neurite outgrowth-induced PC12 cells in differentiation medium supplemented with one of the followings: 0.3% DMSO (n = 9), 50 ng/mL mNGF (n = 9), or 0.3, 3, or 30 µM t-banglene (n = 9 each) (Fig. 1B) using SuperScript VILO (Life Technologies) according to the manufacturer’s instructions. The synthesized cDNA was used as a template for qPCR, which was performed using the SYBR Green real-time PCR Master Mix (TOYOBO, Osaka, Japan). The primers used for RT1 class I and, locus CE1 (RT1-CE1), ATP binding cassette subfamily A member 1 (abca1), myosin light chain 6 (myl6), and hippocampus abundant transcript 1, and β-actin in the rats are listed in Supplementary Table S3. PCR and data analyses were performed using an Applied Biosystems StepOnePlus real-time PCR system (Thermo Fisher Scientific). The relative expression was calculated using the ΔΔCT method. The expression level of each mRNA was normalized to that of rat β-actin.

Statistical Analyses

The results of transcriptome analysis were expressed as means ± standard deviation (S.D.). All other results were expressed as means ± standard error of the mean (S.E.M.). The statistical significance of differences between more than two groups was analyzed using one-way ANOVA or the LPE test per comparison pair. Differences were considered statistically significant at p < 0.05.

RESULTS

t-Banglene Promoted the Neurite Outgrowth of PC12 Cells

To confirm the promotion of neurite outgrowth of PC12 cells by treatment with t-banglene (Fig. 1B), we performed imaging analysis of the treated cells. PC12 cells were incubated in differentiation medium supplemented with 0.3, 3, or 30 µM t-banglene for nine days (Fig. 1B). 0.3% DMSO and 50 ng/mL mNGF were used as negative and positive controls, respectively. The wells were photographed under phase contrast (Figs. 2A–C), and the percentage of PC12 cells with neurites in each image was measured (Fig. 2D). t-Banglene (0.3–30 µM; Fig. 2C, black arrows) and 50 ng/mL mNGF (Fig. 2B, black arrows) strongly induced neurite outgrowth in PC12 cells and significantly increased the percentage of PC12 cells with neurites compared to 0.3% DMSO (p < 0.001, Figs. 2A, D). Thus, we confirmed that t-banglene promoted neurite outgrowth in PC12 cells using our protocol.

Transcriptome Analysis of t-Banglene-Treated PC12 Cells Revealed Four Significant Differentially Expressed Genes

To identify the molecular mechanisms by which t-banglene promotes neurite outgrowth, we performed transcriptome analysis of PC12 cells treated with DMSO or t-banglene and incubated for nine days using comprehensive RNA sequencing. According to the differentially expressed gene analysis, four genes, RT1-CE1, abca1, myl6, and hippocampus abundant transcript 1, exhibited a statistically significant difference in their expression in 3 µM t-banglene-treated PC12 cells compared to 0.3% DMSO (Table 1). The expression of RT-CE1 was upregulated greater than 3.5 fold, whereas that of abca1, myl6l, and hippocampus abundant transcript 1 was respectively downregulated to less than 2.2-, 2.8, or 3.1 folds in 3 µM t-banglene-treated PC12 cells relative to expression in 0.3% DMSO-treated cells (Table 1).

Next, for the result display of the four genes selected by differentially expressed gene analysis, the gene distribution was based on the comparison of expression levels between DMSO- and t-banglene-treated PC12 cells (Supplementary Table S1). These distributions are shown as a scatter plot of read count (FPKM) (Fig. 3A), volume plot of mean expression values (Fig. 3B), and volcano plot of log₂ fold-changes and adjusted p-values (Fig. 3C). Additionally, expression pattern of the four selected genes is shown as a heat map of the one-way hierarchical clustering using Z-score for normalized value (log₂ based) (Fig. 3D). The scatter plot, volume plot, or volcano plot displaying the gene distribution revealed that the expression of RT-CE1 was uniquely upregulated, whereas that of abca1, myl6l, or hippocampus abundant transcript 1 was uniquely downregulated in 3 µM t-banglene-treated PC12 cells relative to expression in 0.3% DMSO-treated cells among all differentially expressed genes (Figs. 3A–C, red dots). The up- or downregulation of the expression of the four genes is also represented as a heat map using spectrum colors (Fig. 3D).

Fig. 3. Transcriptome Analysis of t-Banglene-Treated PC12 Cells Using a Comprehensive RNA Sequencing

Expression levels in 0.3% DMSO- and 3 µM t-banglene-treated PC12 cells (supplementary Table S2) were shown as a scatter plot (A), a volume plot (B), and a volcano plot (C), and four genes, RT1-CE1, abca1, myl6l, and hippocampus abundant transcript 1, selected by statistical analysis of differentially expressed genes, were indicated in these plots (red dots). (A) Scatter plot showed expression levels in DMSO- and t-banglene-treated PC12 cells. X-axis and Y-axis were indicated in normalized values of the t-banglene- and DMSO-treated PC12 cells, respectively. (B) Volume plot showed the log₂ fold-change in expression of DMSO-treated PC12 cells compared to that of expression in t-banglene-treated cells. X-axis: volume, Y-axis: log₂ fold change. (C) Volcano plot showed the log₂ fold change plotting and p-value obtained from the comparison between the expression levels in DMSO- and t-banglene-treated PC12 cells. X-axis: log₂ fold change, Y-axis: -log₁₀ p-value. (D) Result of hierarchical clustering analysis by Euclidean method and complete linkage, which clusters the gene and sample similarity by normalized expression levels (Table 1), shown as a heatmap.

Therefore, transcriptome analysis revealed that t-banglene significantly upregulated RT-CE1 and downregulated abca1, myl6l, and hippocampus abundant transcript 1 expressions in PC12 cells.

t-Banglene Upregulated RT1-CE1 and Downregulated abca1 in PC12 Cells

To confirm the expression levels of the four differentially expressed genes in PC12 cells treated with t-banglene, revealed by transcriptome analysis, we performed the RT-qPCR for these four genes, namely RT1-CE1, abca1, myl6, or hippocampus abundant transcript 1 (Supplementary Table S3). Total RNA samples extracted from PC12 cells treated with one of the following: 0.3% DMSO, 50 ng/mL mNGF, or 0.3, 3, or 30 µM t-banglene and incubated for nine days (Fig. 1B) were used for the RT-qPCR analysis. The expression of RT1-CE1 was significantly upregulated in 3 and 30 µM t-banglene-treated cells (p < 0.001 each) (Fig. 4A), and that of abca1 was significantly downregulated in mNGF (p < 0.05)- and 0.3 (p < 0.05), 3, or 30 µM (p < 0.001 each) t-banglene-treated PC12 cells relative to mRNA expression in 0.3% DMSO-treated cells (Fig. 4B). In contrast, the expression levels of myl6 and hippocampus abundant transcript 1 in t-banglene-treated PC12 cells remained unchanged compared to those in 0.3% DMSO-treated cells (Figs. 4C, D). Single peaks of melt curves in qPCR results of myl6 and hippocampus abundant transcript 1 qPCR were observed using each specific primer (Supplementary Table S3), suggesting that each gene represents a pure single amplicon (data not shown). The other exons in myl6 or hippocampus abundant transcript 1 were amplified by comprehensive RNA sequencing. Therefore, we demonstrated that t-banglene upregulated RT1-CE1 and downregulated abca1 in PC12 cells.

Fig. 4. RT-qPCR Analysis of Four Genes Differentially Expressed in t-Banglene-Treated PC12 Cells

cDNA was synthesized from total RNA of neurite outgrowth-induced PC12 cells in a differentiation medium supplemented with one of the following: 0.3% DMSO (n = 9), 50 ng/mL mNGF (n = 9), or 0.3, 3, or 30 µM t-banglene (n = 9 each). The synthesized cDNA was used as a template for qPCR. Relative expression levels of RT1-CE1 (A), abca1 (B), myl6 (C), or hippocampus abundant transcript 1 (D) were determined by RT-qPCR and normalized to levels of rat β-actin. These expression levels were calculated relative to levels found in DMSO-treated cells (set to 1). Data are represented as means ± S.E.M. of three independent experiments. * p < 0.05, ** p < 0.01, or *** p < 0.001, versus DMSO-treated cells.

Taken together, our results suggest that RT1-CEl upregulation and abca1 downregulation in t-banglene-treated PC12 cells may be linked to the neurite outgrowth-promoting effect.

DISCUSSION

RT1-CEl, similar to major histocompatibility complex class I (MHCI), binds to self or foreign peptides derived from proteolysis of intracellular proteins. It presents these peptides to cytotoxic lymphocytes, which recognize the peptides as self or non-self peptides. This recognition mechanism plays an important role in protecting against bacterial or viral infections and cancer development. In the adult and the developing central nervous system (CNS), MHCI mRNA was found to be expressed by neurons and regulated by kainic acid-activity-dependent plasticity.¹¹⁾ Analyses of MHCI-deficient mice revealed that MHCI is required for the connection refinement between the retina and central targets during development, as well as for long-term potentiation and depression in the hippocampus.¹²⁾ Previous studies have reported that MHCI gene and protein expression plays important roles in neuronal development and function, including in neurite outgrowth,^13,14) synaptic plasticity,^12,15) synaptic specificity,¹⁶⁾ and synapse elimination and learning rules¹⁷⁾ in mature mice or human neurons in the CNS. Additionally, Warre-Cornish et al. reported that MHCI gene and protein expression is increased in neurons with interferon (IFN)-γ-induced neurite outgrowth derived from human induced pluripotent stem cells.¹⁴⁾ We demonstrated that t-banglene upregulated RT1-CE1 in the neurite outgrowth of PC12 cells by transcriptome analysis using comprehensive RNA sequencing (Fig. 3) and RT-qPCR (Fig. 4A). Thus, upregulation of RT1-CEl expression can contribute to the induction of neurite outgrowth in PC12 cells by t-banglene.

ABCA1 is a major cholesterol transporter that regulates the efflux of intracellular cholesterol and phospholipids to apolipoprotein (Apo) A–I, and its mRNA is widely expressed in various organs, including the small intestine, liver, kidney, lung, and brain.¹⁸⁾ In the brain, ApoE is an abundant apolipoprotein, and ABCA1 mediates high density lipoprotein (HDL) cholesterol and ApoE production.¹⁹⁾ ABCA1 dimer interacts with Apo A–I to generate discoidal HDL by removing cellular phospholipid and cholesterol.^20,21) The depletion of cellular cholesterol by β-cyclodextrin decreases the neurite outgrowth in cortical neurons.²²⁾ Therefore, the downregulation of abca1 expression may increases cellular cholesterols to reduce HDL formation from Apo A–I by removing cellular cholesterols. Additionally, the downregulation of abca1 expression is observed upon exposure to pro-inflammatory cytokines, interleukin-1β,²³⁾ IFN-γ,²⁴⁾ tumor necrosis factor-α,²⁵⁾ or their mixture,²⁶⁾ in which LXR-dependent or -independent pathways are reported to be involved. Interestingly, these cytokines also possess neurotrophic activity, including neurite outgrowth promoting effect.^14,27–31) We demonstrated that t-banglene downregulates abca1 expression in the neurite outgrowth of PC12 cells using transcriptome analysis (Fig. 3) and RT-qPCR (Fig. 4B). Taken together, t-banglene may induce neurite outgrowth in PC12 cells by abca1 downregulation via an increase of cellular cholesterols to reduce HDL formation and a pro-inflammatory activation.

Previous studies have shown that various concentrations, such as 10 and 30 µM, 0.03, 0.3, and 3 µM, or 1 µM, of t-banglene promoted neurite outgrowth in PC12 cells, primary-cultured rat cortical neurons, and hfNSCs; however, at the concertation 0.03 µM, t-banglene did not increase neurite number or improve viability.^1,2,4) Thus, we examined neurite outgrowth in PC12 cells treated with 0.3, 3, and 30 µM t-banglene using our protocol and confirmed that t-banglene promoted neurite outgrowth in PC12 cells at all tested concentrations (Fig. 2). Furthermore, we performed transcriptome analysis on PC12 cells treated with 3 µM t-banglene and employed qPCR to assess the expression of specific genes in PC12 cells treated with 0.3, 3, or 30 µM t-banglene.

However, in Fig. 4, 0.3 µM t-banglene did not upregulate RT1-CE1 expression in PC12 cells, and the degree of significant abcal downregulation in 0.3 µM t-banglene-treated PC12 cells was low relative to that which promoted neurite outgrowth. The discrepancy between neurite outgrowth and RT1-CE1/abca1 expression in 0.3 µM t-banglene-treated PC12 cells may suggest that upon treatment with 0.3 µM t-banglene, there is a time lag between promotion of neurite outgrowth and altered RT1-CE1/abca1 expression in PC12 cells, or it may indicate no direct relationship between them, or suggest that altered RT1-CE1/abca1 expression is an outcome of neurite outgrowth. Thus, our data indicate that RT1-CE1 and ABCA1 are good candidate genes for regulators of t-banglene mediated neurite outgrowth. Further research, including gene-knockdown experiments using specific siRNAs and/or protein expression analysis by Western blotting remains necessary to clarify the precise mechanisms regulated by RT1-CE1 and ABCA1 in the neurite outgrowth stimulated by t-banglene.

Taken together, our data suggest that alteration of RT1-CE1 and ABCA1 gene expression by t-banglene contribute to its promotion of neurite outgrowth, suggesting that these two genes are good candidate targets for modulating neurite outgrowth mediated by t-banglene. In conclusion, the findings in this study elucidate, for the first time, the molecular mechanisms underlying neurotrophic activity of t-banglene in PC12 cells.

Acknowledgments

This work was supported by the Tokushima Bunri University Grant for Educational Reform and Collaborative Research Grants TBU2016-2-3 and TBU2020-2-1 (to M. S.) and the Japan Society for the Promotion of Science (JSPS), Grants-in-Aid for Scientific Research (C) 18K06645 (to T. K.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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