Amorfrutin B Compromises Hypoxia/Ischemia-induced Activation of Human Microglia in a PPARγ-dependent Manner: Effects on Inflammation, Proliferation Potential, and Mitochondrial Status.

Amorfrutin B is a selective PPARγ modulator that we demonstrated to be a promising neuroprotective compound in cellular models of stroke and perinatal asphyxia. Although neuronal mechanisms of amorfrutin B-evoked neuroprotection have been identified, none of them reflects the actions of the compound on microglia, which play a pivotal role in brain response to hypoxia/ischemia. Here, we provide evidence for amorfrutin B-induced effects on human microglia subjected to hypoxia/ischemia; the compound counteracts inflammation, and influences mitochondrial status and proliferation potential in a PPARγ-dependent manner. Post-treatment with amorfrutin B decreased the IBA1 fluorescence intensity, reduced caspase-1 activity, and downregulated IL1B/IL-1ß and TNFA but not IL10/IL-10 expression, which was upregulated. Amorfrutin B also stimulated PPARγ signaling, as evidenced by increased mRNA and/or protein levels of PPARγ and PGC1α. In addition, amorfrutin B reversed the hypoxia/ischemia-evoked effects on mitochondria-related parameters, such as mitochondrial membrane potential, BCL2/BCL2 expression and metabolic activity, which were correlated with diminished proliferation potential of microglia. Interestingly, the inhibitory effect of amorfrutin B on the proliferation potential and mitochondrial function of microglia is opposite to the stimulatory effect of amorfrutin B on mouse neuronal survival, as evidenced by increased neuronal viability and reduced neurodegeneration. In summary, this study showed for the first time that amorfrutin B compromises hypoxia/ischemia-induced activation of human microglia in a PPARγ-dependent manner, which involves inhibiting inflammation, normalizing mitochondrial status, and controlling proliferation potential. These data extend the protective potential of amorfrutin B in the pharmacotherapy of hypoxic/ischemic brain injury, targeting not only neurons but also activated microglia.

Posttreatment with PaPE-1 Protects from Aß-Induced Neurodegeneration Through Inhibiting the Expression of Alzheimer's Disease-Related Genes and Apoptosis Process That Involves Enhanced DNA Methylation of Specific Genes.

Targeting the non-nuclear estrogen receptor (ER) signaling has been postulated as novel therapeutic strategy for central nervous system pathologies. Recently, we showed that newly designed PaPE-1 (Pathway Preferential Estrogen-1), which selectively activates ER non-nuclear signaling pathways, elicited neuroprotection in a cellular model of Alzheimer's disease (AD) when it was applied at the same time as amyloid-ß (Aß). Since delayed treatment reflects clinical settings better than cotreatment does, current basic study proposes a novel therapeutic approach for AD that relies on a posttreatment with PaPE-1. In this study, mouse neuronal cell cultures treated with preaggregated Aß1-42 (10 µM) showed the presence of extracellular Aß1-42, confirming the adequacy of the AD model used. We are the first to demonstrate that a 24-h delayed posttreatment with PaPE-1 decreased the degree of Aß-induced neurodegeneration, restored neurite outgrowth, and inhibited the expression of AD-related genes, i.e., Rbfox, Apoe, Bace2, App, and Ngrn, except for Chat, which was stimulated. In addition, PaPE-1 elicited anti-apoptotic effects by inhibiting Aß-induced caspase activities as well as attenuating apoptotic chromatin condensation, and in these ways, PaPE-1 prevented neuronal cell death. Posttreatment with PaPE-1 also downregulated the Aß-affected mRNA expression of apoptosis-specific factors, such as Bax, Gsk3b, Fas, and Fasl, except for Bcl2, which was upregulated by PaPE-1. In parallel, PaPE-1 decreased the protein levels of BAX, FAS, and FASL, which were elevated in response to Aß. PaPE-1 elicited a decrease in the BAX/BCL2 ratio that corresponds to increased methylation of the Bax gene. However, the PaPE-1-evoked Bcl2 gene hypermethylation suggests other PaPE-1-dependent mechanisms to control Aß-induced apoptosis.

Involvement of the peroxisome proliferator-activated receptor gamma (Pparγ) and matrix metalloproteinases-2 and -9 (Mmp-2 and -9) in the mechanism of action of di(2-ethylhexyl)phthalate (DEHP) in cultured mouse brain astrocytes and neurons.

Di(2-ethylhexyl)phthalate (DEHP) is one of the most widely used phthalates in industry. It has been shown that, after entering the body, DEHP has the ability to cross the blood-placenta and blood-brain barriers. One of the proposed mechanisms of action of DEHP is the activation of peroxisome proliferator-activated receptors (PPARs). Many different functions of PPARγ in cells have been demonstrated, one of which is the modulation of the activation of matrix metalloproteinases (MMPs). The aim of this study was to investigate the role of Pparγ, Mmp-2, and Mmp-9 in the mechanism of action of DEHP. The experiments were performed on in vitro primary murine neurons and astrocytes. The results showed that DEHP has a pro-apototic effect on neurons, causing an increase in caspase-3 activity and in the number of apoptotic bodies. However, in astrocytes, the increase in caspase-3 activity was not related to the apoptosis process, as no increase in the formation of apoptotic bodies was observed. Moreover, DEHP increased the proliferation of astrocytes, which was confirmed by the increase in the amount and expression of the Ki-67 protein. In astrocytes, DEHP decreased the expression of the Pparγ and Mmp-9 proteins but increased the expression of the Mmp-2 protein. In DEHP neurons, it increased the expression of the Pparγ protein but decreased the expression of the Mmp-2 and Mmp-9 proteins.

Posttreatment with Ospemifene Attenuates Hypoxia- and Ischemia-Induced Apoptosis in Primary Neuronal Cells via Selective Modulation of Estrogen Receptors.

Stroke and perinatal asphyxia have detrimental effects on neuronal cells, causing millions of deaths worldwide each year. Since currently available therapies are insufficient, there is an urgent need for novel neuroprotective strategies to address the effects of cerebrovascular accidents. One such recent approach is based on the neuroprotective properties of estrogen receptors (ERs). However, activation of ERs by estrogens may contribute to the development of endometriosis or hormone-dependent cancers. Therefore, in this study, we utilized ospemifene, a novel selective estrogen receptor modulator (SERM) already used in dyspareunia treatment. Here, we demonstrated that posttreatment with ospemifene in primary neocortical cell cultures subjected to 18 h of hypoxia and/or ischemia followed by 6 h of reoxygenation has robust neuroprotective potential. Ospemifene partially reverses hypoxia- and ischemia-induced changes in LDH release, the degree of neurodegeneration, and metabolic activity. The mechanism of the neuroprotective actions of ospemifene involves the inhibition of apoptosis since the compound decreases caspase-3 overactivity during hypoxia and enhances mitochondrial membrane potential during ischemia. Moreover, in both models, ospemifene decreased the levels of the proapoptotic proteins BAX, FAS, FASL, and GSK3ß while increasing the level of the antiapoptotic protein BCL2. Silencing of specific ERs showed that the neuroprotective actions of ospemifene are mediated mainly via ESR1 (during hypoxia and ischemia) and GPER1 (during hypoxia), which is supported by ospemifene-evoked increases in ESR1 protein levels in hypoxic and ischemic neurons. The results identify ospemifene as a promising neuroprotectant, which in the future may be used to treat injuries due to brain hypoxia/ischemia.

Pre-reproductive stress in adolescent female rats alters maternal care and DNA methylation patterns across generations.

Stress during development affects maternal behavior and offspring phenotypes. Stress in adolescence is particularly consequential on brain development and maturation, and is implicated in several psychiatric disorders. We previously showed that pre-reproductive stress (PRS) in female adolescent rats affects behavior and corticotropin releasing hormone receptor 1 (CRHR1) expression in first- (F1) and second- (F2) generation offspring. We further showed that offspring phenotypes are partially reversed by post-stress treatment with fluoxetine (FLX) or the CRHR1 antagonist NBI27914 (NBI). Epigenetic processes, such as DNA methylation, are implicated in the stress response and interact with maternal care quality across generations. Here, we asked whether PRS and FLX or NBI exposure would affect maternal care and global DNA methylation in the brains of exposed dams and their adult F1 and paternally-derived F2 offspring. We found that PRS decreased self-care while increasing pup-care behaviors. PRS also increased DNA methylation in the amygdala of dams and their F1 male offspring, but decreased it in F2 females. Drug treatment had no effect on maternal care, but affected DNA methylation patterns in F0 and F1 generations. Furthermore, PRS altered the expression of DNA methylating enzymes in brain, blood and oocytes. Finally, maternal care variables differentially predicted methylation levels in PRS and control offspring. Thus, the effects of adolescent stress are long-lasting and impact methylation levels across three generations. Combined with our findings of epigenetic changes in PRS-exposed oocytes, the present data imply that biological changes and social mechanisms act in concert to influence adult offspring phenotypes.

Emerging Evidence on Membrane Estrogen Receptors as Novel Therapeutic Targets for Central Nervous System Pathologies.

Nuclear- and membrane-initiated estrogen signaling cooperate to orchestrate the pleiotropic effects of estrogens. Classical estrogen receptors (ERs) act transcriptionally and govern the vast majority of hormonal effects, whereas membrane ERs (mERs) enable acute modulation of estrogenic signaling and have recently been shown to exert strong neuroprotective capacity without the negative side effects associated with nuclear ER activity. In recent years, GPER1 was the most extensively characterized mER. Despite triggering neuroprotective effects, cognitive improvements, and vascular protective effects and maintaining metabolic homeostasis, GPER1 has become the subject of controversy, particularly due to its participation in tumorigenesis. This is why interest has recently turned toward non-GPER-dependent mERs, namely, mERα and mERß. According to available data, non-GPER-dependent mERs elicit protective effects against brain damage, synaptic plasticity impairment, memory and cognitive dysfunctions, metabolic imbalance, and vascular insufficiency. We postulate that these properties are emerging platforms for designing new therapeutics that may be used in the treatment of stroke and neurodegenerative diseases. Since mERs have the ability to interfere with noncoding RNAs and to regulate the translational status of brain tissue by affecting histones, non-GPER-dependent mERs appear to be attractive targets for modern pharmacotherapy for nervous system diseases.

Amorfrutin B Protects Mouse Brain Neurons from Hypoxia/Ischemia by Inhibiting Apoptosis and Autophagy Processes Through Gene Methylation- and miRNA-Dependent Regulation.

Amorfrutin B is a selective modulator of the PPARγ receptor, which has recently been identified as an effective neuroprotective compound that protects brain neurons from hypoxic and ischemic damage. Our study demonstrated for the first time that a 6-h delayed post-treatment with amorfrutin B prevented hypoxia/ischemia-induced neuronal apoptosis in terms of the loss of mitochondrial membrane potential, heterochromatin foci formation, and expression of specific genes and proteins. The expression of all studied apoptosis-related factors was decreased in response to amorfrutin B, both during hypoxia and ischemia, except for the expression of anti-apoptotic BCL2, which was increased. After post-treatment with amorfrutin B, the methylation rate of the pro-apoptotic Bax gene was inversely correlated with the protein level, which explained the decrease in the BAX/BCL2 ratio as a result of Bax hypermethylation. The mechanisms of the protective action of amorfrutin B also involved the inhibition of autophagy, as evidenced by diminished autophagolysosome formation and the loss of neuroprotective properties of amorfrutin B after the silencing of Becn1 and/or Atg7. Although post-treatment with amorfrutin B reduced the expression levels of Becn1, Nup62, and Ambra1 during hypoxia, it stimulated Atg5 and the protein levels of MAP1LC3B and AMBRA1 during ischemia, supporting the ambiguous role of autophagy in the development of brain pathologies. Furthermore, amorfrutin B affected the expression levels of apoptosis-focused and autophagy-related miRNAs, and many of these miRNAs were oppositely regulated by amorfrutin B and hypoxia/ischemia. The results strongly support the position of amorfrutin B among the most promising anti-stroke and wide-window therapeutics.

Prenatal Exposure to Triclocarban Impairs ESR1 Signaling and Disrupts Epigenetic Status in Sex-Specific Ways as Well as Dysregulates the Expression of Neurogenesis- and Neurotransmitter-Related Genes in the Postnatal Mouse Brain.

Triclocarban is a highly effective and broadly used antimicrobial agent. Humans are continually exposed to triclocarban, but the safety of prenatal exposure to triclocarban in the context of neurodevelopment remains unknown. In this study, we demonstrated for the first time that mice that had been prenatally exposed to environmentally relevant doses of triclocarban had impaired estrogen receptor 1 (ESR1) signaling in the brain. These mice displayed decreased mRNA and protein expression levels of ESR1 as well as hypermethylation of the Esr1 gene in the cerebral cortex. Prenatal exposure to triclocarban also diminished the mRNA expression of Esr2, Gper1, Ahr, Arnt, Cyp19a1, Cyp1a1, and Atg7, and the protein levels of CAR, ARNT, and MAP1LC3AB in female brains and decreased the protein levels of BCL2, ARNT, and MAP1LC3AB in male brains. In addition, exposure to triclocarban caused sex-specific alterations in the methylation levels of global DNA and estrogen receptor genes. Microarray and enrichment analyses showed that, in males, triclocarban dysregulated mainly neurogenesis-related genes, whereas, in females, the compound dysregulated mainly neurotransmitter-related genes. In conclusion, our data identified triclocarban as a neurodevelopmental risk factor that particularly targets ESR1, affects apoptosis and autophagy, and in sex-specific ways disrupts the epigenetic status of brain tissue and dysregulates the postnatal expression of neurogenesis- and neurotransmitter-related genes.

Is the commonly used UV filter benzophenone-3 a risk factor for the nervous system?

Benzophenone-3 (2-hydroxy-4-methoxybenzophenone, oxybenzone, or BP-3) is one of the most frequently used UV radiation absorbents, which are commonly referred to as sunscreen filters. Its widespread use in industrial applications provides protection against the photodegradation of a wide range of products but at the same time creates the risk of human exposure to benzophenone-3 unbeknownst to the individuals exposed. Topically applied benzophenone-3 penetrates individual skin layers, enters the bloodstream, and is excreted in the urine. In addition, benzophenone-3 easily crosses the placental barrier, which creates the risk of exposure to this substance in the prenatal period. Despite the widespread use and occurrence of benzophenone-3 in the human environment, little knowledge of the mechanisms underlying the effect of benzophenone-3 on the nervous system was available until recently. Only the most recent research, including studies by our group, has enabled the identification of new molecular mechanisms through which benzophenone-3 affects embryonic neuronal cells and the developing mammalian brain. Benzophenone-3 has been shown to induce neurotoxicity and apoptotic processes and inhibit autophagy in embryonic neuronal cells. Benzophenone-3 also alters expression and impairs function of receptors necessary for the proper development and function of the nervous system. The most worrying finding seems to be that benzophenone-3 contributes to an increased risk of developmental abnormalities and/or epigenetically based degeneration of neuronal cells by changing the epigenetic status of neuronal cells.

Post-Treatment with Amorfrutin B Evokes PPARγ-Mediated Neuroprotection against Hypoxia and Ischemia.

In this study, we demonstrate for the first time that amorfrutin B, a selective modulator of peroxisome proliferator-activated receptor gamma-PPARγ, can protect brain neurons from hypoxia- and ischemia-induced degeneration when applied at 6 h post-treatment in primary cultures. The neuroprotective effect of amorfrutin B suggests that it promotes mitochondrial integrity and is capable of inhibiting reactive oxygen species-ROS activity and ROS-mediated DNA damage. PPARγ antagonist and Pparg mRNA silencing abolished the neuroprotective effect of amorfrutin B, which points to agonistic action of the compound on the respective receptor. Interestingly, amorfrutin B stimulated the methylation of the Pparg gene, both during hypoxia and ischemia. Amorfrutin B also increased the protein level of PPARγ during hypoxia but decreased the mRNA and protein levels of PPARγ during ischemia. Under ischemic conditions, amorfrutin B-evoked hypermethylation of the Pparg gene is in line with the decrease in the mRNA and protein expression of PPARγ. However, under hypoxic conditions, amorfrutin B-dependent hypermethylation of the Pparg gene does not explain the amorfrutin B-dependent increase in receptor protein expression, which suggests other regulatory mechanisms. Other epigenetic parameters, such as HAT and/or sirtuins activities, were affected by amorfrutin B under hypoxic and ischemic conditions. These properties position the compound among the most promising anti-stroke and wide-window therapeutics.

Selective Targeting of Non-nuclear Estrogen Receptors with PaPE-1 as a New Treatment Strategy for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial and severe neurodegenerative disorder characterized by progressive memory decline, the presence of Aß plaques and tau tangles, brain atrophy, and neuronal loss. Available therapies provide moderate symptomatic relief but do not alter disease progression. This study demonstrated that PaPE-1, which has been designed to selectively activate non-nuclear estrogen receptors (ERs), has anti-AD capacity, as evidenced in a cellular model of the disease. In this model, the treatment of mouse neocortical neurons with Aß (5 and 10 µM) induced apoptosis (loss of mitochondrial membrane potential, activation of caspase-3, induction of apoptosis-related genes and proteins) accompanied by increases in levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) as well as reduced cell viability. Following 24 h of exposure, PaPE-1 inhibited Aß-evoked effects, as shown by reduced parameters of neurotoxicity, oxidative stress, and apoptosis. Because PaPE-1 downregulated Aß-induced Fas/FAS expression but upregulated that of Aß-induced FasL, the role of PaPE-1 in controlling the external apoptotic pathway is controversial. However, PaPE-1 normalized Aß-induced loss of mitochondrial membrane potential and restored the BAX/BCL2 ratio, suggesting that the anti-AD capacity of PaPE-1 particularly relies on inhibition of the mitochondrial apoptotic pathway. These data provide new evidence for an anti-AD strategy that utilizes the selective targeting of non-nuclear ERs with PaPE-1.

Autophagy-related neurotoxicity is mediated via AHR and CAR in mouse neurons exposed to DDE.

DDE (dichlorodiphenyldichloroethylene) is an environmental metabolite of the pesticide DDT, which is still present in the environment, and its insecticidal properties are used to fight malaria and the Zika virus disease. We showed for the first time that the neurotoxic effects of DDE involve autophagy, as demonstrated by elevated levels of Becn1, Map1lc3a/MAP1LC3A, Map1lc3b, and Nup62/NUP62 and an increase in autophagosome formation. The suggestion that the aryl hydrocarbon receptor (AHR) and the constitutive androstane receptor (CAR) are involved in the neurotoxic effect of DDE was supported by increases in the mRNA and protein expression of these receptors, as detected by qPCR, ELISA, immunofluorescence labeling and confocal microscopy. Selective antagonists of the receptors, including alpha-naphthoflavone, CH223191, and CINPA 1, inhibited p,p'-DDE- and o,p'-DDE-induced LDH release and caspase-3 activity, while specific siRNAs (Ahr and Car siRNA) reduced the levels of p,p'-DDE- and o,p'-DDE-induced autophagosome formation. Although the neurotoxic effects of DDE were isomer independent, the mechanisms of p,p'- and o,p'-DDE were isomer specific. Therefore, we identified previously unknown mechanisms of the neurotoxic actions of DDE that, in addition to inducing apoptosis, stimulate autophagy in mouse neocortical cultures and induce AHR and CAR signaling.

Glutathione Deficiency and Alterations in the Sulfur Amino Acid Homeostasis during Early Postnatal Development as Potential Triggering Factors for Schizophrenia-Like Behavior in Adult Rats.

Impaired glutathione (GSH) synthesis and dopaminergic transmission are important factors in the pathophysiology of schizophrenia. Our research aimed to assess the effects of l-buthionine-(S,R)-sulfoximine (BSO), a GSH synthesis inhibitor, and GBR 12909, a dopamine reuptake inhibitor, administered alone or in combination, to Sprague-Dawley rats during early postnatal development (p5-p16), on the levels of GSH, sulfur amino acids, global DNA methylation, and schizophrenia-like behavior. GSH, methionine (Met), homocysteine (Hcy), and cysteine (Cys) contents were determined in the liver, kidney, and in the prefrontal cortex (PFC) and hippocampus (HIP) of 16-day-old rats. DNA methylation in the PFC and HIP and schizophrenia-like behavior were assessed in adulthood (p90-p93). BSO caused the tissue-dependent decreases in GSH content and alterations in Met, Hcy, and Cys levels in the peripheral tissues and in the PFC and HIP. The changes in these parameters were accompanied by alterations in the global DNA methylation in the studied brain structures. Parallel to changes in the global DNA methylation, deficits in the social behaviors and cognitive functions were observed in adulthood. Only BSO + GBR 12909-treated rats exhibited behavioral alterations resembling positive symptoms in schizophrenia patients. Our results suggest the usefulness of this neurodevelopmental model for research on the pathomechanism of schizophrenia.

Correction to: the Effects of Exposure to Mephedrone During Adolescence on Brain Neurotransmission and Neurotoxicity in Adult Rats.

Acknowledgments: This study was supported by the Grant No 2013/09/B/NZ7/04104 from the National Science Center (Poland).

Effects of exposure to 5-MeO-DIPT during adolescence on brain neurotransmission and neurotoxicity in adult rats.

PURPOSE: Tryptamine hallucinogen 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a serotonin transporter inhibitor with high affinity for serotonin 5-HT1A and 5-HT2A/C receptors. We showed previously that 5-MeO-DIPT in a single dose increased neurotransmitter release in brain regions of rats and elicited single- and double-strand DNA breaks. Herein we investigated the effects of repeated-intermittent 5-MeO-DIPT administration in adolescence on dopamine (DA), serotonin (5-HT) and glutamate release in brain regions of adult rats. Furthermore, we examined caspase-3 activity, oxidative DNA damage, the Gpx3, Sod1, Ht1a and Ht2a mRNA expression levels, and cell viability. METHODS: Neurotransmitter release was measured by microdialysis in freely moving animals. Caspase-3 activity was assessed colorimetrically, and oxidative DNA damage with the comet assay, while the Gpx3, Sod1, Ht1a and Ht2a mRNA expression levels were assessed by real-time polymerase chain reaction. Cell viability was studied in SH-SY5Y and Hep G2 cells by the MTT test. RESULTS: We observed changed responses of DA, 5-HT and glutamate neurons to a challenge dose of 5-MeO-DIPT when animals were treated repeatedly in adolescence with this hallucinogen. The basal extracellular levels of DA and 5-HT were decreased in the striatum and nucleus accumbens, while glutamate level was increased in the nucleus accumbens and frontal cortex. The damage of cortical DNA, increased Gpx3 and Sod1 mRNA expression and affected caspase-3 activity were also observed. Furthermore, decreased Ht1a and Ht2a mRNA expression in the frontal cortex and marked cytotoxicity of 5-MeO-DIPT were found. CONCLUSIONS: These results suggest that 5-MeO-DIPT given repeatedly during adolescence affects brain neurotransmission and shows neurotoxic potential observed in adult animals.

Triclosan-Evoked Neurotoxicity Involves NMDAR Subunits with the Specific Role of GluN2A in Caspase-3-Dependent Apoptosis.

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitising products. A number of studies have shown the presence of TCS in different human tissues such as blood, adipose tissue, the liver, brain as well as in breast milk and urine. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are widely expressed in the central nervous system and which play key roles in excitatory synaptic transmission. There is, however, no data on the involvement of NMDAR subunits in the apoptotic and neurotoxic effects of TCS. Our experiments are the first to show that TCS used at environmentally relevant concentrations evoked NMDA-dependent effects in neocortical neurons in primary cultures, as MK-801, an uncompetitive NMDA receptor antagonist, reduced the levels of TCS-induced ROS production as well as caspase-3 activity and LDH release. TCS caused a decrease in protein expression of all the studied NMDA receptor subunits (GluN1, GluN2A, GluN2B) that were measured at 3, 6 and 24 h post-treatment. However, at 48 h of the experiment, the level of the GluN1 subunit returned to the control level, and the levels of the other subunits showed a tendency to increase. In TCS-treated neocortical cells, protein profiles of NMDAR subunits measured up to 24 h were similar to mRNA expression of GluN1 and GluN2A, but not to GluN2B mRNA. In this study, cells transiently transfected with GluN1, GluN2A or GluN2B siRNA exhibited reduced levels of LDH release, which suggests the involvement of all of the studied NMDAR subunits in the neurotoxic action of TCS. According to our data, GluN1 and GluN2A were mainly responsible for neuronal cell death as evidenced by neutral red uptake, whereas GluN2A was involved in TCS-induced caspase-3-dependent apoptosis. We suggest that TCS-evoked apoptosis and neurotoxicity could be related to transient degradation of NMDAR subunits in mouse neurons. Furthermore, recycling of NMDAR subunits in response to TCS is possible. Because transfections with specific siRNA did not completely abolish the effects of TCS as compared to cells transfected with negative siRNA in this study, other NMDAR-independent mechanisms of TCS action are also possible.

Prenatal Exposure to Benzophenone-3 Impairs Autophagy, Disrupts RXRs/PPARγ Signaling, and Alters Epigenetic and Post-Translational Statuses in Brain Neurons.

The UV absorber benzophenone-3 (BP-3) is the most extensively used chemical substance in various personal care products. Despite that BP-3 exposure is widespread, knowledge about the impact of BP-3 on the brain development is negligible. The present study aimed to explore the mechanisms of prenatal exposure to BP-3 in neuronal cells, with particular emphasis on autophagy and nuclear receptors signaling as well as the epigenetic and post-translational modifications occurring in response to BP-3. To observe the impact of prenatal exposure to BP-3, we administered BP-3 to pregnant mice, and next, we isolated brain tissue from pretreated embryos for primary cell neocortical culture. Our study revealed that prenatal exposure to BP-3 (used in environmentally relevant doses) impairs autophagy in terms of BECLIN-1, MAP1LC3B, autophagosomes, and autophagy-related factors; disrupts the levels of retinoid X receptors (RXRs) and peroxisome proliferator-activated receptor gamma (PPARγ); alters epigenetic status (i.e., attenuates HDAC and sirtuin activities); inhibits post-translational modifications in terms of global sumoylation; and dysregulates expression of neurogenesis- and neurotransmitter-related genes as well as miRNAs involved in pathologies of the nervous system. Our study also showed that BP-3 has good permeability through the BBB. We strongly suggest that BP-3-evoked effects may substantiate a fetal basis of the adult onset of neurological diseases, particularly schizophrenia and Alzheimer's disease.

The Effects of Exposure to Mephedrone During Adolescence on Brain Neurotransmission and Neurotoxicity in Adult Rats.

According to the European Drug Report (2016), the use of synthetic cathinones, such as mephedrone, among young people has rapidly increased in the last years. Studies in humans indicate that psychostimulant drug use in adolescence increases risk of drug abuse in adulthood. Mephedrone by its interaction with transporters for dopamine (DAT) and serotonin (SERT) stimulates their release to the synaptic cleft. In animal studies, high repeated doses of mephedrone given to adolescent but not adult mice or rats induced toxic changes in 5-hydroxytryptamine (5-HT) neurons. The aim of our study was to investigate the effects of mephedrone given in adolescence on brain neurotransmission and possible neuronal injury in adult rats. Adolescent male rats were given mephedrone (5 mg/kg) for 8 days. In vivo microdialysis in adult rats showed an increase in dopamine (DA), 5-HT, and glutamate release in the nucleus accumbens and frontal cortex but not in the striatum in response to challenge dose in animals pretreated with mephedrone in adolescence. The 5-HT and 5-hydroxyindoleacetic acid contents decreased in the striatum and nucleus accumbens while DA turnover rates were decreased in the striatum and nucleus accumbens. The oxidative damage of DNA assessed with the alkaline comet assay was found in the cortex of adult rats. Therefore, the administration of repeated low doses of mephedrone during adolescence does not seem to induce injury to 5-HT and DA neurons. The oxidative stress seems to be responsible for possible damage of cortical cell bodies which causes maladaptive changes in serotonergic and dopaminergic neurons.

Prenatal exposure to benzophenone-3 (BP-3) induces apoptosis, disrupts estrogen receptor expression and alters the epigenetic status of mouse neurons.

Current evidence indicates that benzophenone-3 (BP-3) can pass through the placental and blood-brain barriers and thus can likely affect infant neurodevelopment. Despite widespread exposure, data showing the effects of BP-3 on the developing nervous system are scarce. This study revealed for the first time that prenatal exposure to BP-3 led to apoptosis and neurotoxicity, altered the levels of estrogen receptors (ERs) and changed the epigenetic status of mouse neurons. In the present study, subcutaneous injections of pregnant mice with BP-3 at 50 mg/kg, which is an environmentally relevant dose, evoked activation of caspase-3 and lactate dehydrogenase (LDH) release as well as substantial loss of mitochondrial membrane potential in neocortical cells of their embryonic offspring. Apoptosis-focused microarray analysis of neocortical cells revealed up-regulation of 22 genes involved in apoptotic cell death. This effect was supported by increased BAX and CASP3 mRNA and protein levels, as evidenced by qPCR, ELISAs and western blots. BP-3-induced apoptosis and neurotoxicity were accompanied by decreases in the mRNA and protein expression levels of ESR1 and ESR2 (also known as ERα and ERß), with a simultaneous increase in GPER1 (also known as GPR30) expression. In addition to the demonstration that treatment of pregnant mice with BP-3 induced apoptosis, caused neurotoxicity and altered ERs expression levels in neocortical cells of their embryonic offspring, we showed that prenatal administration of BP-3 inhibited global DNA methylation as well as reduced DNMTs activity. BP-3 also caused specific hypomethylation of the genes Gper1 and Bax, an effect that was accompanied by increased mRNA and protein expression levels. In addition, BP-3 caused hypermethylation of the genes Esr1, Esr2 and Bcl2, which could explain the reduced mRNA and protein levels of the estrogen receptors. This study demonstrated for the first time that prenatal exposure to BP-3 caused severe neuronal apoptosis that was accompanied by impaired ESR1/ESR2 expression, enhanced GPER1 expression, global DNA hypomethylation and altered methylation statuses of apoptosis-related and ERs genes. We suggest that the effects of BP-3 in embryonic neurons may be the fetal basis of the adult onset of nervous system disease.

Benzophenone-3 Impairs Autophagy, Alters Epigenetic Status, and Disrupts Retinoid X Receptor Signaling in Apoptotic Neuronal Cells.

Benzophenone-3 (BP-3) is the most widely used compound among UV filters for the prevention of photodegradation. Population studies have demonstrated that it penetrates through the skin and crosses the blood-brain barrier. However, little is known about the impact of BP-3 on the nervous system and its possible adverse effects on the developing brain. We demonstrated that the neurotoxic effects of BP-3 were accompanied by the induction of apoptosis, as evidenced by apoptosis-related caspase-3 activation and apoptotic body formation as well as the inhibition of autophagy, as determined by the downregulation of autophagy-related genes, decreased autophagosome formation, and reduced LC3B-to-LC3A ratio. In this study, we showed for the first time that the BP-3-induced apoptosis of neuronal cells is mediated via the stimulation of RXRα signaling and the attenuation of RXRß/RXRγ signaling, as demonstrated using selective antagonist and specific siRNAs as well as by measuring the mRNA and protein expression levels of the receptors. This study also demonstrated that environmentally relevant concentrations of BP-3 were able to inhibit autophagy and disrupt the epigenetic status of neuronal cells, as evidenced by the inhibition of global DNA methylation as well as the reduction of histone deacetylases and histone acetyl transferases activity, which may increase the risks of neurodevelopmental abnormalities and/or neural degenerations.