Chlorpyrifos inhibits neural induction via Mfn1-mediated mitochondrial dysfunction in human induced pluripotent stem cells

Organophosphates, such as chlorpyrifos (CPF), are widely used as insecticides in agriculture. CPF is known to induce cytotoxicity, including neurodevelopmental toxicity. However, the molecular mechanisms of CPF toxicity at early fetal stage have not been fully elucidated. In this study, we examined the mechanisms of CPF-induced cytotoxicity using human induced pluripotent stem cells (iPSCs). We found that exposure to CPF at micromolar levels decreased intracellular ATP levels. As CPF suppressed energy production that is a critical function of the mitochondria, we focused on the effects of CPF on mitochondrial dynamics. CPF induced mitochondrial fragmentation via reduction of mitochondrial fusion protein mitofusin 1 (Mfn1) in iPSCs. In addition, CPF reduced the expression of several neural differentiation marker genes in iPSCs. Moreover, knockdown of Mfn1 gene in iPSCs downregulated the expression of PAX6, a key transcription factor that regulates neurogenesis, suggesting that Mfn1 mediates neural induction in iPSCs. Taken together, these results suggest that CPF induces neurotoxicity via Mfn1-mediated mitochondrial fragmentation in iPSCs. Thus, mitochondrial dysfunction in iPSCs could be used as a possible marker for cytotoxic effects by chemicals.

Scientific RepoRts | 7:40925 | DOI: 10.1038/srep40925 Morphological changes of mitochondria are known to contribute to homeostasis 14,15 . Under normal circumstances, mitochondria fuses together and forms excessive tubular networks (mitochondrial fusion). These fusion is regulated by fusion factors mitofusin 1 and 2 (Mfn1,Mfn2) and optic atrophy 1 (Opa1) 16,17 . In contrast, under stress conditions, mitochondrial networks convert into large numbers of small fragments with spherical and punctate morphology (mitochondrial fission), and are regulated by fission factors, such as fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) 18,19 . This morphological dynamics contributes to the maintenance of mitochondrial functions, including energy generation 14 . Moreover, several studies have shown the relationship between mitochondrial fragmentation and cellular and neurodevelopmental defects. For example, Mfn1 or Mfn2 knockout mice die in midgestation embryo, accompanying with developmental delay. In addition, embryonic fibroblasts from these knockout mice display distinct types of fragmented mitochondria, a phenotype due to a severe reduction in mitochondrial fusion 20 . Thus, Mfn1 is considered to be functionally different from Mfn2. In support to this, Mfn1, not Mfn2, is reported to contribute to Opa1-mediated fusion of mitochondrial inner membrane 16 .
In the present study, we investigated the effect of CPF on neural differentiation using human induced pluripotent stem cells (iPSCs) as a model of human organ development. We focused on the effects of micromolar levels of CPF on mitochondrial dynamics, examining the molecular mechanisms of the process. Our results show that micromolar CPF levels inhibited ATP production through Mfn1 reduction, followed by mitochondrial fragmentation. Moreover, Mfn1-mediated mitochondrial dysfunction suppressed early neural induction by decreasing levels of PAX6, a key transcription factor that regulates neurogenesis. These data suggest that CPF-induced neurodevelopmental toxicity is based on impairment of mitochondrial functions in human iPSCs.

Effect of CPF on neural differentiation of iPSCs.
To investigate whether CPF affects early neurodevelopment, we examined neural differentiation capability of iPSCs, which was induced by dual SMAD inhibition protocol 21 (Fig. 1A). First, we determined the critical CPF concentration, affecting neural differentiation. At day 4 after neural induction with different concentrations of CPF, the expression of PAX6, an early neuroectodermal marker that regulates neurogenesis 22 , was analyzed using real-time PCR. We found that exposure to 30 μ M CPF significantly decreased PAX6 gene expression (Fig. 1B). Next, we performed time course experiments for expression of several neural differentiation markers at days 2, 4, 6, and 8 after exposure to 30 μ M CPF. At day 9, almost all cells exposed by CPF (30 μ M) were detached from the culture dish. Real-time PCR analysis revealed upregulated expression of PAX6 by day 4, and FOXG1, a neuroectodermal marker that also regulates neurogenesis 23 , thereafter ( Fig. 1C and D). Representative neural maturation marker NCAM1 24 continuously increased, confirming that further neural differentiation occurred (Fig. 1E). In addition, CPF exposure reduced the expression of these neural induction markers by day 6 ( Fig. 1C-E). These data suggest that CPF has an inhibitory effect on early neural differentiation of iPSCs.
Mitochondrial function of iPSCs exposed to CPF. As neural differentiation process requires ATP as a source of energy 25 , we examined intracellular ATP content in iPSCs. Treatment with 30 μ M CPF significantly reduced the ATP content of the cells ( Fig. 2A). We have previously shown that 0.1 μ M carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which functions as a mitochondrial uncoupler 26 , decreased ATP levels in iPSCs. Because CPF inhibited ATP production, we focused on several mitochondrial functions. Mitochondrial membrane potential (MMP) was decreased by exposure to 30 μ M CPF for 24 h ( Fig. 2B and C). As a positive control, exposure to 0.1 μ M CCCP reduced MMP ( Figure S1). In addition, CPF exposure increased the number of cells with fragmented mitochondria displaying punctate morphology (Fig. 2D) and decreased the number of cells exhibiting mitochondrial fusion (Fig. 2E). We have already confirmed that 0.1 μ M CCCP also increased the occurrence of fragmented mitochondria. These results suggest that CPF induces mitochondrial dysfunction, including MMP depolarization and mitochondrial fragmentation, in iPSCs.
Expression of mitochondrial fission and fusion factors in iPSCs exposed to CPF. To examine the molecular mechanisms by which CPF induces mitochondrial fragmentation in iPSCs, we assessed the expression levels of mitochondrial fission (Fis1 and Drp1) and fusion genes (Mfn1, Mfn2, and OPA1). Real-time PCR analysis showed that the gene expression of the factors was not altered after CPF exposure (Fig. 3A). Interestingly, western blot analysis revealed that CPF significantly decreased Mfn1 protein levels. In contrast, protein expression levels of other factors, including Mfn2, were not changed ( Fig. 3B and C). These data suggest that CPF-induced mitochondrial fragmentation is caused by reduction of Mfn1 protein levels.

Effects of CPF in iPSC-derived neural progenitor cells.
To investigate whether the effects of CPF selectively occur in the early stage of neural differentiation in iPSCs, we used iPSC-derived neural progenitor cells (NPCs), which were induced by dual SMAD inhibition protocol 21 ( Figure S1A). Treatment with 30 μ M CPF had little effect on ATP content ( Figure S1B). Similarly, exposure to 30 μ M CPF had little effect on mitochondrial morphology ( Figure S1C and D), which was confirmed by the fact that CPF did not alter the protein levels of mitochondrial fission and fusion factors containing Mfn1 ( Figure S1E). These data suggest that iPSCs, not NPCs, are sensitive to CPF exposure.

Effect of Mfn1 knockdown on neural induction of iPSCs. To further investigate the involvement of
Mfn1 in the effects of CPF on neural induction, we performed knockdown (KD) of Mfn1, using lentivirus-delivered shRNAs. Real-time PCR analysis showed that KD was selective for Mfn1, not Mfn2, and that the efficiency was approximately 70% (Fig. 4A). The KD effects were also confirmed by protein levels ( Fig. 4B and C). The Mfn1 KD cells were used to perform neural induction. Real-time PCR analysis revealed that Mfn1 KD decreased the expression of PAX6 (day 4), FOXG1 (day 6) and NCAM1 (day 6) (Fig. 4D). These data suggest that Mfn1 is involved in CPF-mediated negative effects on neural induction of iPSCs.

Negative regulation of neural induction by CPF exposure. A previous report indicates that ERK
signaling inhibits neural induction via PAX6 silencing in human embryonic stem cells 27 . ERK has been reported to be activated after depletion of Mfn1 28 . We focused on ERK signaling in the effect of CPF on neural induction. We found that CPF exposure significantly increased basal ERK phosphorylation levels, which were abolished by treatment with the ERK inhibitor U0126 (Fig. 5A and B). To further study whether PAX6 downregulation in CPF-exposed cells occurred through ERK signaling, we examined the effect of U0126 on PAX6 expression. Incubation with U0126 recovered the expression levels of PAX6 (Fig. 5C). These data suggest that CPF activates ERK and prevents neural induction via PAX6 downregulation.

Effect of Mfn1 knockdown on neural induction.
To confirm the involvement of Mfn1 in the inhibition of neural induction by CPF, we used Mfn1 KD cells. Mfn1 KD significantly increased basal ERK phosphorylation levels that were abolished by treatment with the ERK inhibitor U0126 (Fig. 6A and B). To further study whether PAX6 downregulation in Mfn1 KD cells occurred through ERK signaling, we examined the effect of U0126 on PAX6 expression. Mfn1 KD decreased PAX6 by 64% by in the vehicle-treated cells. In contrast, Mfn1 KD decreased PAX6 by 30% in the U0126-treated cells. Thus, incubation with U0126 partially recovered the PAX6 expression in the Mfn1 KD cells (Fig. 6C). Taken together, these data suggest that Mfn1 reduction by CPF exposure activates ERK and prevents neural induction via PAX6 downregulation.

Discussion
In the present study, we demonstrated that exposure to micromolar CPF targeted mitochondrial quality control in human iPSCs. We showed that CPF induced Mfn1 reduction, thereby promoting mitochondrial fragmentation. These negative effects of CPF on mitochondrial quality control could suppress ATP production and neural differentiation. Based on the data observed in our study, Fig. 7 shows a proposed mechanism of CPF cytotoxicity via mitochondrial dysfunction.
Our studies showed that treatment with micromolar CPF levels caused mitochondrial dysfunction of human iPSCs (Fig. 2). We observed that iPSCs were sensitive to CPF exposure, unlike iPSC-derived NPCs ( Figure S1). Previous reports support this difference in CPF sensitivity. The inhibitory effect of CPF on DNA synthesis in undifferentiated C6 glioma cells is found to be much higher than in differentiated cells 29 . In vivo studies indicate that immature organisms are more susceptible to CPF-induced toxicity compared to adults due to lower levels of CPF metabolizing enzymes 30 . Thus, the difference in CPF sensitivity between iPSCs and NPCs may be dependent on the maturation of CPF detoxification pathways. We are currently conducting experiments to determine the mechanism causing the differences in sensitivity to CPF.
We showed that CPF induced mitochondrial fragmentation via Mfn1 reduction (Figs 2 and 3). Consistent with this, our previous knockdown studies indicated that Mfn1 reduction was sufficient to promote mitochondrial dysfunction 31 . CPF-induced Mfn1 reduction might mediate mitochondrial fragmentation, decrease ATP levels, and inhibit iPSC growth. Although Mfn2 is also involved in mitochondrial fission and energy supply processes 32,33 , our results indicated that CPF specifically targeted Mfn1, not Mfn2. Regarding this apparent CPF specificity, E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5) has been reported to selectively bind to Mfn1 dependent on its acetylation, and degrade among all mitochondrial proteins, including Mfn2 34 . In addition, we have reported that organotin compounds induced Mfn1 degradation through MARCH5, We demonstrated that ERK phosphorylation mediated the negative effects of CPF on early neural differentiation (Figs 1, 4 and 5). A previous report indicates that Mfn1 directly binds Ras and Raf, resulting in the inhibition of Ras-Raf-ERK signaling by the biochemical analysis 35,36 . Mfn1 reduction by CPF or shRNA may reverse this ERK signaling inhibition. Mobilization of Ca 2+ from intracellular stores, including mitochondria was reported to result in phosphorylation of MAPKs, as the process was suppressed by chelation of intracellular Ca 2+ in human T lymphoblastoid cells 37 . As mitochondria are known to uptake into the matrix of any Ca 2+ that has accumulated in the cytosol, dependent on MMP 38 , mitochondrial dysfunction by CPF exposure may cause an overload of Ca 2+ , resulting in ERK activation. Moreover, ERK signaling was reported to inhibit neural induction by PAX6 silencing via upregulation of stemness factors NANOG/OCT4 and downregulation of homeobox transcription factor OTX2 27 . NANOG and OCT4 act as repressors of PAX6 induction, whereas OTX2 is a positive inducer of PAX6 27 . Therefore, ERK signaling evoked by CPF could affect the expression of these transcriptional network, including NANOG, OCT4 and OTX2, by regulating PAX6. In future studies, we should further investigate the mechanisms of CPF-induced negative regulation of neural induction via ERK. We further demonstrated that Mfn1 reduction mediated cytotoxic effects of CPF on iPSCs via PAX6 downregulation (Figs 5 and 6). FOXG1 was downregulated, along with PAX6, during neural differentiation of iPSCs exposed to CPF. PAX6 and FOXG1 act as transcriptional regulators during forebrain development in vertebrates 39,40 . Targeted disruption of PAX6 and FOXG1 in rodents led to the loss of anterior neural tissues, suggesting the central role of these genes in forebrain development 41,42 . CPF causes various defects in the development of hippocampus and cortex of rodents 43 . Thus, CPF-induced defects of forebrain architecture may be caused by transcriptional silencing of anterior neural markers during early neurogenesis. As NCAM1 was downregulated during neural differentiation of iPSCs exposed to CPF, further studies using NPCs are required to reveal how CPF affects neural maturation processes.
In summary, our results demonstrate a novel mechanism underlying cytotoxicity, including neurodevelopmental toxicity of CPF in iPSCs. Recently, significant progress has been made in the induction of differentiation of pluripotent stem cells into a variety of cell types 44 . Further studies are needed to evaluate the developmental effects of CPF on various types of iPSC-derived cells. Moreover, we show that CPF toxicity is caused by Mfn1-mediated mitochondrial dysfunction, which is involved in the cytotoxicity of organotin compounds 31 . Thus, mitochondrial functions influenced by Mfn1 might be a good starting point for investigating toxic mechanisms induced by exposure to other chemicals.  medium was Neural maintenance medium [NMM; a 1∶ 1 mixture of DMEM/F12 (Thermo Fisher Scientific) and Neurobasal (Thermo Fisher Scientific) containing N2 (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA; Thermo Fisher Scientific), 2-ME, PS]. For passage, NPCs were dissociated into single cells using Accumax and cultured in NMM supplemented with EGF (20 ng/ml), FGF2 (20 ng/ml) and Y-27632.
Neural differentiation procedure. For the induction of neuronal lineages, dual SMAD inhibition protocol was used as previously described 21 with modifications. Briefly, iPSC colonies were dissociated into single cells with Accumax. The cells were seeded at a density of 7 × 10 4 cells/cm 2 in TeSR-E8 medium on Matrigel-coated plates in order to reach nearly confluent within two days after seeding. The initial differentiation medium was knockout serum replacement (KSR) medium [Knockout DMEM (Thermo Fisher Scientific) containing KSR (Thermo Fisher Scientific), L-glutamine, NEAA, 2-ME, PS] with SB431542 (TGFβ inhibitor, 10 μ M) and LDN193189 (BMP inhibitor, 1 μ g/ml). After 4 days, N2 medium [Neurobasal containing N2, B27, GlutaMAX, PS] was added to the KSR medium with LDN193189 every two days.

Measurement of intracellular ATP levels. Intracellular ATP content was measured using an ATP
Determination Kit (Thermo Fisher Scientific), according to the manufacturer's protocol. Briefly, the cells were washed and lysed with 0.1% Triton X-100/PBS. The resulting cell lysates were added to a reaction mixture containing 0.5 mM D-luciferin, 1 mM DTT, and 1.25 μ g/mL luciferase and incubated for 30 min at room temperature. Luminescence was measured using a Fluoroskan Ascent FL microplate reader (Thermo Fisher Scientific). The luminescence intensities were normalized to the total protein content.

Measurement of MMP.
A Cell Meter JC-10 Mitochondrial Membrane Potential Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) was used to detect MMP. Briefly, the cells were suspended in staining buffer containing JC-10 and incubated for 20 min at room temperature. After the cells were treated with CPF, a FACS Aria II cell sorter (BD Biosciences) was used to measure the fluorescence intensity ratio, JC-aggregate (F-590)/JC-monomer (F-535).
Assessment of mitochondrial fusion. After treatment with CPF (30 μ M, 72 h), the cells were fixed with 4% paraformaldehyde and stained with 50 nM MitoTracker Red CMXRos (Cell Signaling Technology, Danvers, MA, USA) and 5 μ g/mL Hoechst 33342 (Sigma-Aldrich). Changes in mitochondrial morphology were observed using a confocal laser microscope (Nikon A1). Images (n = 5) of random fields were taken, and the number of cells displaying mitochondrial fusion (< 10% punctiform) was determined in each image, as previously reported 46 .
Scientific RepoRts | 7:40925 | DOI: 10.1038/srep40925 of 8 μ g/mL hexadimethrine bromide (Sigma-Aldrich) for 24 h. After medium exchange, the cells were subjected to selection with 1 μ g/mL puromycin for 24 h and cultured for an additional 72 h prior to functional analyses. Statistical analysis. All data are presented as means ± standard deviation (SD). Analysis of variance (ANOVA) followed by post-hoc Bonferroni test was used to analyze data in Figs 1, 3C, 4, 5, and 6. Student's t test was used to analyze data in Figs 2, 3A, S1, and S2. P-values < 0.05 were considered statistically significant.