Neuroprotective effect of 3,3'-Diindolylmethane against perinatal asphyxia involves inhibition of the AhR and NMDA signaling and hypermethylation of specific genes.

Each year, 1 million children die due to perinatal asphyxia; however, there are no effective drugs to protect the neonatal brain against hypoxic/ischemic damage. In this study, we demonstrated for the first time the neuroprotective capacity of 3,3'-diindolylmethane (DIM) in an in vivo model of rat perinatal asphyxia, which has translational value and corresponds to hypoxic/ischemic episodes in human newborns. Posttreatment with DIM restored the weight of the ipsilateral hemisphere and normalized cell number in the brain structures of rats exposed to perinatal asphyxia. DIM also downregulated the mRNA expression of HIF1A-regulated Bnip3 and Hif1a which is a hypoxic marker, and the expression of miR-181b which is an indicator of perinatal asphyxia. In addition, DIM inhibited apoptosis and oxidative stress accompanying perinatal asphyxia through: downregulation of FAS, CASP-3, CAPN1, GPx3 and SOD-1, attenuation of caspase-9 activity, and upregulation of anti-apoptotic Bcl2 mRNA. The protective effects of DIM were accompanied by the inhibition of the AhR and NMDA signaling pathways, as indicated by the reduced expression levels of AhR, ARNT, CYP1A1, GluN1 and GluN2B, which was correlated with enhanced global DNA methylation and the methylation of the Ahr and Grin2b genes. Because our study provided evidence that in rat brain undergoing perinatal asphyxia, DIM predominantly targets AhR and NMDA, we postulate that compounds that possess the ability to inhibit their signaling are promising therapeutic tools to prevent stroke.

Triclocarban impairs autophagy in neuronal cells and disrupts estrogen receptor signaling via hypermethylation of specific genes.

Although an increasing body of evidence suggests that triclocarban, a phenyl ether classified as a contaminant of emerging concern, presents a risk to development, there is limited data available on the potential interplay of triclocarban with the developing mammalian nervous system. This study was aimed to investigate the impact of environmentally pervasive chemical triclocarban on autophagy and estrogen receptor-mediated signaling pathways in mouse neurons. The study showed that triclocarban impaired autophagy and disrupted estrogen receptor signaling in mouse embryonic neurons in primary culture. Triclocarban used at environmentally relevant concentrations inhibited the mRNA and protein expression of ESR1 and GPER1 but not ESR2. The triclocarban-induced decrease in the expression of estrogen receptors was supported by the colocalization of the receptors in mouse neurons and corresponded to hypermethylation of the Esr1 and Gper1 genes. Selective antagonists increased the effects of triclocarban, which suggests that the neurotoxic effects of triclocarban, in addition to decreasing estrogen receptor expression, are mediated via inhibition of the neuroprotective capacity of the receptors. Furthermore, Becn1 and Atg7 siRNAs potentiated the caspase-3-dependent effect of triclocarban, which points to triclocarban-induced impairment of autophagy. Indeed, triclocarban dysregulated the expression of autophagy-related genes, and caused a time-dependent inhibition of the mRNA expression of Becn1, Map1lc3a, Map1lc3b, Nup62, and Atg7, which was correlated with a decrease in the protein levels of MAP1LC3B, BECN1 and autophagosomes, but not NUP62 protein level which was increased. Intriguingly, the Esr1 and Gper1 siRNAs did not affect the level of autophagosomes, suggesting that the triclocarban-induced impairment of autophagy is independent of the triclocarban-induced disruption of estrogen receptor signaling in mammalian neurons. Because our data provided evidence that triclocarban has the capacity to impair autophagy and disrupt estrogen receptor signaling in brain neurons at an early developmental stage, we postulate to categorize the compound as a neurodevelopmental risk factor.

The neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy that depends on HDAC and AhR/CYP1A1 but not ERα/CYP19A1 signaling.

There are no studies examining the effects of 3,3'-diindolylmethane (DIM) in neuronal cells subjected to ischemia. Little is also known about the roles of apoptosis and autophagy as well as AhR and ERα signaling and HDACs in DIM action. We demonstrated for the first time the strong neuroprotective capacity of DIM in mouse primary hippocampal cell cultures exposed to ischemia at early and later stages of neuronal development. The protective effects of DIM were mediated via inhibition of ischemia-induced apoptosis and autophagy that was accompanied by a decrease in AhR/CYP1A1 signaling and an increase in HDAC activity. DIM decreased the levels of pro-apoptotic factors, i.e., Fas, Caspase-3, and p38 mitogen-activated protein kinase (MAPK). DIM also reduced the protein levels of autophagy-related Beclin-1 (BECN1) and microtubule-associated proteins 1A/1B light chain (LC3), partially reversed the ischemia-induced decrease in Nucleoporin 62 (NUP62) and inhibited autophagosome formation. In addition, DIM completely reversed the ischemia-induced decrease in histone deacetylase (HDAC) activity in hippocampal neurons. Although DIM inhibited AhR/CYP1A1 signaling, it did not influence the protein expression levels of ERα and ERα-regulated CYP19A1 which are known to be controlled by AhR. This study demonstrated for the first time, that the neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy and depends on AhR/CYP1A1 signaling and HDAC activity, thus creating the possibility of developing new therapeutic strategies that target neuronal degeneration at specific molecular levels.

Triclocarban Disrupts the Epigenetic Status of Neuronal Cells and Induces AHR/CAR-Mediated Apoptosis.

Triclocarban is a phenyl ether that has recently been classified as a contaminant of emerging concern. Evidence shows that triclocarban is present in human tissues, but little is known about the impact of triclocarban on the nervous system, particularly at early developmental stages. This study demonstrated that triclocarban that was used at environmentally relevant concentrations induced apoptosis in mouse embryonic neurons, inhibited sumoylation, and changed the epigenetic status, as evidenced by impaired activities of HDAC, sirtuins, and DNMT, global DNA hypomethylation, and alterations of methylation levels of bax, bcl2, Ahr, and Car genes. The use of selective antagonists and specific siRNAs, which was followed by the co-localization of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) in mouse neurons, points to the involvement of AHR and CAR in triclocarban-induced neurotoxicity. A 24-h treatment with triclocarban enhanced protein levels of the receptors which was paralleled by Car hypomethylation and Ahr hypermethylation. Car hypomethylation is in line with global DNA hypomethylation and explains the increased mRNA and protein levels of CAR in response to triclocarban. Ahr hypermethylation could reflect reduced Ahr mRNA expression and corresponds to lowered protein levels after 3- and 6-h exposures to triclocarban that is likely related to proteasomal degradation of activated AHR. We hypothesize that the triclocarban-induced apoptosis in mouse neurons and the disruption of epigenetic status involve both AHR- and CAR-mediated effects, which may substantiate a fetal basis of the adult onset of neurological diseases; however, the expression of the receptors is regulated in different ways.

Apoptosis Induced by the UV Filter Benzophenone-3 in Mouse Neuronal Cells Is Mediated via Attenuation of Erα/Pparγ and Stimulation of Erß/Gpr30 Signaling.

Although benzophenone-3 (BP-3) has frequently been reported to play a role in endocrine disruption, there is insufficient data regarding the impact of BP-3 on the nervous system, including its possible adverse effects on the developing brain. Our study demonstrated that BP-3 caused neurotoxicity and activated apoptosis via an intrinsic pathway involving the loss of mitochondrial membrane potential and the activation of caspases-9 and -3 and kinases p38/MAPK and Gsk3ß. These biochemical alterations were accompanied by ROS production, increased apoptotic body formation and impaired cell survival, and by an upregulation of the genes involved in apoptosis. The BP-3-induced effects were tissue-specific and age-dependent with the most pronounced effects observed in neocortical cells at 7 days in vitro. BP-3 changed the messenger RNA (mRNA) expression levels of Erα, Erß, Gpr30, and Pparγ in a time-dependent manner. At 3 h of exposure, BP-3 downregulated estrogen receptor mRNAs but upregulated Pparγ mRNA. After prolonged exposures, BP-3 downregulated the receptor mRNAs except for Erß mRNA that was upregulated. The BP-3-induced patterns of mRNA expression measured at 6 and 24 h of exposure reflected alterations in the protein levels of the receptors and paralleled their immunofluorescent labeling. Erα and Pparγ agonists diminished, but Erß and Gpr30 agonists stimulated the BP-3-induced apoptotic and neurotoxic effects. Receptor antagonists caused the opposite effects, except for ICI 182,780. This is in line with a substantial reduction in the effects of BP-3 in cells with siRNA-silenced Erß/Gpr30 and the maintenance of BP-3 effects in Erα- and Pparγ siRNA-transfected cells. We showed for the first time that BP-3-affected mRNA and protein expression levels of Erα, Erß, Gpr30, and Pparγ, paralleled BP-3-induced apoptosis and neurotoxicity. Therefore, we suggest that BP-3-evoked apoptosis of neuronal cells is mediated via attenuation of Erα/Pparγ and stimulation of Erß/Gpr30 signaling.

Bazedoxifene and raloxifene protect neocortical neurons undergoing hypoxia via targeting ERα and PPAR-γ.

Selective estrogen receptor modulators (SERMs) such as bazedoxifene and raloxifene are recognized to mainly act via estrogen receptors (ERs), but there is no study examining the involvement of PPAR-γ in their actions, especially in neurons undergoing hypoxia. Little is also known about age-dependent actions of the SERMs on neuronal tissue challenged with hypoxia. In this study, bazedoxifene and raloxifene protected neocortical cells against hypoxia at early and later developmental stages. Both SERMs evoked caspase-3-independent neuroprotection and increased protein levels of ERα (66 and 46 kDa isoforms) and PPAR-γ. In addition, bazedoxifene enhanced expression of ERα-regulated Cyp19a1 mRNA. Using double siRNA silencing, for the first time we demonstrated a key role of ERα and PPAR-γ in the neuroprotective action of the SERMs in neocortical neurons undergoing hypoxia. This study provides prospects for the development of a new therapeutic strategies against hypoxic brain injury that selectively target ERα and/or PPAR-γ.

Selective Aryl Hydrocarbon Receptor Modulator 3,3'-Diindolylmethane Impairs AhR and ARNT Signaling and Protects Mouse Neuronal Cells Against Hypoxia.

The neuroprotective potential of 3,3'-diindolylmethane (DIM), which is a selective aryl hydrocarbon receptor modulator, has recently been shown in cellular and animal models of Parkinson's disease and lipopolysaccharide-induced inflammation. However, there are no data concerning the protective capacity and mechanisms of DIM action in neuronal cells exposed to hypoxia. The aim of the present study was to investigate the neuroprotective potential of DIM against the hypoxia-induced damage in mouse hippocampal cells in primary cultures, with a particular focus on DIM interactions with the aryl hydrocarbon receptor (AhR), its nuclear translocator ARNT, and estrogen receptor ß (ERß). In the present study, 18 h of hypoxia induced apoptotic processes, in terms of the mitochondrial membrane potential, activation of caspase-3, and fragmentation of cell nuclei. These effects were accompanied by substantial lactate dehydrogenase release and neuronal cell death. The results of the present study demonstrated strong neuroprotective and anti-apoptotic actions of DIM in hippocampal cells exposed to hypoxia. In addition, DIM decreased the Ahr and Arnt mRNA expression and stimulated Erß mRNA expression level. DIM-induced mRNA alterations were mirrored by changes in protein levels, except for ERß, as detected by ELISA, Western blotting, and immunofluorescence labeling. We also demonstrated that DIM decreased the expression of AhR-regulated CYP1A1. Using specific siRNAs, we provided evidence that impairment of AhR and ARNT, but not ERß plays a key role in the neuroprotective action of DIM against hypoxia-induced cell damage. This study may have implication for identifying new agents that could protect neurons against hypoxia by targeting AhR/ARNT signaling.

The Crucial Involvement of Retinoid X Receptors in DDE Neurotoxicity.

Dichlorodiphenyldichloroethylene (DDE) is a primary environmental and metabolic degradation product of the pesticide dichlorodiphenyltrichloroethane (DDT). It is one of the most toxic compounds belonging to organochlorines. DDE has never been commercially produced; however, the parent pesticide DDT is still used in some developing countries for disease-vector control of malaria. DDT and DDE remain in the environment because these chemicals are resistant to degradation and bioaccumulate in the food chain. Little is known, however, about DDE toxicity during the early stages of neural development. The results of the present study demonstrate that DDE induced a caspase-3-dependent apoptosis and caused the global DNA hypomethylation in mouse embryonic neuronal cells. This study also provided evidence for DDE-isomer-non-specific alterations of retinoid X receptor α (RXRα)- and retinoid X receptor ß (RXRß)-mediated intracellular signaling, including changes in the levels of the receptor mRNAs and changes in the protein levels of the receptors. DDE-induced stimulation of RXRα and RXRß was verified using selective antagonist and specific siRNAs. Co-localization of RXRα and RXRß was demonstrated using confocal microscopy. The apoptotic action of DDE was supported at the cellular level through Hoechst 33342 and calcein AM staining experiments. In conclusion, the results of the present study demonstrated that the stimulation of RXRα- and RXRß-mediated intracellular signaling plays an important role in the propagation of DDE-induced apoptosis during early stages of neural development.

RXRα, PXR and CAR xenobiotic receptors mediate the apoptotic and neurotoxic actions of nonylphenol in mouse hippocampal cells.

In the present study, we investigated the role of the retinoid X receptor (RXR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the apoptotic and toxic effects of nonylphenol in mouse primary neuronal cell cultures. Our study demonstrated that nonylphenol activated caspase-3 and induced lactate dehydrogenase (LDH) release in hippocampal cells, which was accompanied by an increase in the mRNA expression and protein levels of RXRα, PXR and CAR. Nonylphenol stimulated Rxra, Pxr, and Car mRNA expression. These effects were followed by increase in the protein levels of particular receptors. Immunofluorescence labeling revealed the cellular distribution of RXRα, PXR and CAR in hippocampal neurons in response to nonylphenol, shortening of neurites and cytoplasmic shrinking, as indicated by MAP2 staining. It also showed NP-induced translocation of receptor-specific immunofluorescence from cytoplasm to the nucleus. The use of specific siRNAs demonstrated that Rxra-, Pxr-, and Car-siRNA-transfected cells were less vulnerable to nonylphenol-induced activation of caspase-3 and LDH, thus confirming the key involvement of RXRα/PXR/CAR signaling pathways in the apoptotic and neurotoxic actions of nonylphenol. These new data give prospects for the targeting xenobiotic nuclear receptors to protect the developing nervous system against endocrine disrupting chemicals.

Neuroprotective action of raloxifene against hypoxia-induced damage in mouse hippocampal cells depends on ERα but not ERß or GPR30 signalling.

Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERß) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18 h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erß and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erß mRNA expression level were in parallel with ERß immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERß or GPR30 in neuroprotective action of raloxifene against hypoxia-induced cell damage. This study may have implications for the treatment or prevention of hypoxic brain injury and the administration of current or new generations of SERMs specific to ERα. This article is part of a Special Issue entitled "Sex steroids and brain disorders".

Apoptotic and neurotoxic actions of 4-para-nonylphenol are accompanied by activation of retinoid X receptor and impairment of classical estrogen receptor signaling.

4-para-Nonylphenol (NP) is a non-ionic surfactant that has widespread and uncontrolled distribution in the environment. Little is known, however, about its actions on neuronal cells during critical developmental periods. This study aimed to investigate the mechanisms underlying the apoptotic and toxic actions of NP on mouse embryonic neuronal cells and the possible interactions of NP with estrogen receptor (ER)- and retinoid X receptor (RXR)-mediated intracellular signaling. Treatment of mouse hippocampal neuronal cell cultures with NP (5 and 10µM) induced apoptotic and neurotoxic effects. The 2 and 7 day-old mouse hippocampal cultures were vulnerable to 5 and 10µM NP, whereas 12 day-old cultures responded only to the highest concentration of NP, thus suggesting an age-dependent action of the chemical on neuronal cells. The use of specific inhibitors did not support the involvement of calpains in NP-induced apoptosis, but indicated caspase-8- and caspase-9-dependent effects of NP. Specific ER antagonists MPP and PHTPP potentiated the NP-induced loss of mitochondrial membrane potential and increase in lactate dehydrogenase (LDH) release whereas, ER agonists PPT and DPN inhibited these effects. RXR antagonist HX531 diminished the NP-evoked loss of mitochondrial membrane potential, the activity of caspase-3 and LDH release. In addition, exposure to NP inhibited ERα- and ERß-specific immunofluorescence but stimulated RXR-specific immunolabeling in mouse hippocampal cells. In conclusion, our study demonstrated that the apoptotic and toxic actions of NP on neuronal cells in early development is accompanied by an impairment of ER- and stimulation of RXR-mediated signaling pathways. Taking into account NP-induced alterations in mRNA expression levels of particular types of RXRs, we suggest that NP affected mainly RXRα and RXRß, but not RXRγ signaling.

Isomer-nonspecific action of dichlorodiphenyltrichloroethane on aryl hydrocarbon receptor and G-protein-coupled receptor 30 intracellular signaling in apoptotic neuronal cells.

Extended residual persistence of the pesticide dichlorodiphenyltrichloroethane (DDT) raises concerns about its long-term neurotoxic effects. Little is known, however, about DDT toxicity during the early stages of neural development. This study demonstrated that DDT-induced apoptosis of mouse embryonic neuronal cells is a caspase-9-, caspase-3-, and GSK-3ß-dependent process, which involves p,p'-DDT-specific impairment of classical ERs. It also provided evidence for DDT-isomer-nonspecific alterations of AhR- and GPR30-mediated intracellular signaling, including changes in the levels of the receptor and receptor-regulated mRNAs, and also changes in the protein levels of the receptors. DDT-induced stimulation of AhR-signaling and reduction of GPR30-signaling were verified using selective ligands and specific siRNAs. Co-localization of the receptors was demonstrated with confocal microscopy, and the presence of functional GPR30 was detected by electrophysiology. This study demonstrates that stimulation of AhR-signaling and impairment of GPR30-signaling play important roles in the propagation of DDT-induced apoptosis during the early stages of neural development.

The key involvement of estrogen receptor ß and G-protein-coupled receptor 30 in the neuroprotective action of daidzein.

Phytoestrogens have received considerable attention because they provide an array of beneficial effects, such as neuroprotection. To better understand the molecular and functional link between phytoestrogens and classical as well as membrane estrogen receptors (ERs), we investigated the effect of daidzein on the glutamate-mediated apoptotic pathway. Our study demonstrated that daidzein (0.1-10µM) inhibited the pro-apoptotic and neurotoxic effects caused by glutamate treatment. Hippocampal, neocortical and cerebellar tissues responded to the inhibitory action of daidzein on glutamate-activated caspase-3 and lactate dehydrogenase (LDH) release in a similar manner. Biochemical data were supported at the cellular level by Hoechst 33342 and calcein AM staining. The sensitivity of neuronal cells to daidzein-mediated protection was most prominent in hippocampal cultures at an early stage of development 7th day in vitro. A selective estrogen receptor ß (ERß) antagonist, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5,-a]pyrimidin-3-yl]phenol (PHTPP), and a selective G-protein-coupled receptor 30 (GPR30) antagonist, 3aS(∗),4R(∗),9bR(∗))-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G15), reversed the daidzein-mediated inhibition of glutamate-induced loss of membrane mitochondrial potential, caspase-3 activity, and LDH release. A selective ERα antagonist, methyl-piperidino-pyrazole (MPP), did not influence any anti-apoptotic effect of daidzein. However, a high-affinity estrogen receptor antagonist, 7α,17ß-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI) 182,780, and a selective GPR30 agonist, (±)-1-[(3aR(∗),4S(∗),9bS(∗))-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone (G1), intensified the protective action of daidzein against glutamate-induced loss of membrane mitochondrial potential and LDH release. In siRNA ERß- and siRNA GPR30-transfected cells, daidzein did not inhibit the glutamate-induced effects. Twenty-four hour exposure to glutamate did not affect the cellular distribution of ERß and GPR30, but caused greater than 100% increase in the levels of the receptors. Co-treatment with daidzein decreased the level of ERß without significant changing of the GPR30 protein level. Here, we elucidated neuroprotective effects of daidzein at low micromolar concentrations and demonstrated that the phytoestrogens may exert their effects through novel extranuclear GPR30 and the classical transcriptionally acting ERß. These studies uncover key roles of the ERß and GPR30 intracellular signaling pathways in mediating the anti-apoptotic action of daidzein and may provide insight into new strategies to treat or prevent neural degeneration.