ERRα regulates synaptic transmission through reactive oxygen species in hippocampal neurons.

Reactive oxygen species (ROS) play multiple roles in synaptic transmission, and estrogen-related receptor α (ERRα) is involved in regulating ROS production. The purpose of our study was to explore the underlying effect of ERRα on ROS production, neurite formation and synaptic transmission. Our results revealed that knocking down ERRα expression affected the formation of neuronal neurites and dendritic spines, which are the basic structures of synaptic transmission and play important roles in learning, memory and neuronal plasticity; moreover, the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were decreased. These abnormalities were reversed by overexpression of human ERRα. Additionally, we also found that knocking down ERRα expression increased intracellular ROS levels in neurons. ROS inhibitor PBN rescued the changes in neurite formation and synaptic transmission induced by ERRα knockdown. These results indicate a new possible cellular mechanism by which ERRα affects intracellular ROS levels, which in turn regulate neurite and dendritic spine formation and synaptic transmission.

GSDMD knockdown attenuates phagocytic activity of microglia and exacerbates seizure susceptibility in TLE mice.

BACKGROUND: Temporal lobe epilepsy (TLE) is often characterized pathologically by severe neuronal loss in the hippocampus. Phagocytic activity of microglia is essential for clearing apoptotic neuronal debris, allowing for repair and regeneration. Our previous research has shown that gasdermin D (GSDMD)-mediated pyroptosis is involved in the pathogenesis of TLE. However, whether GSDMD-mediated pyroptosis influences the accumulation of apoptotic neurons remains unclear. Therefore, the present study was designed to investigate whether phagocytic activity of microglia is involved in GSDMD-mediated pyroptosis and the pathogenesis of TLE. METHODS: To establish a TLE model, an intra-amygdala injection of kainic acid (KA) was performed. The Racine score and local field potential (LFP) recordings were used to assess seizure severity. Neuronal death in the bilateral hippocampus was assessed by Nissl staining and TUNEL staining. Microglial morphology and phagocytic activity were detected by immunofluorescence and verified by lipopolysaccharide (LPS) and the P2Y12R agonist 2MeSADP. RESULTS: GSDMD knockdown augmented the accumulation of apoptotic neurons and seizure susceptibility in TLE mice. Microglia activated and transition to the M1 type with increased pro-inflammatory cytokines. Furthermore, GSDMD knockdown attenuated the migration and phagocytic activity of microglia. Of note, LPS-activated microglia attenuated seizure susceptibility and the accumulation of apoptotic neurons in TLE after GSDMD knockdown. A P2Y12R selective agonist, 2MeSADP, enhanced the migration and phagocytic activity of microglia. CONCLUSIONS: Our results demonstrate that GSDMD knockdown exacerbates seizure susceptibility and the accumulation of apoptotic neurons by attenuating phagocytic activity of microglia. These findings suggest that GSDMD plays a protective role against KA-induced seizure susceptibility.

KCTD13-mediated ubiquitination and degradation of GluN1 regulates excitatory synaptic transmission and seizure susceptibility.

Temporal lobe epilepsy (TLE) is the most common and severe form of epilepsy in adults; however, its underlying pathomechanisms remain elusive. Dysregulation of ubiquitination is increasingly recognized to contribute to the development and maintenance of epilepsy. Herein, we observed for the first time that potassium channel tetramerization domain containing 13 (KCTD13) protein, a substrate-specific adapter for cullin3-based E3 ubiquitin ligase, was markedly down-regulated in the brain tissue of patients with TLE. In a TLE mouse model, the protein expression of KCTD13 dynamically changed during epileptogenesis. Knockdown of KCTD13 in the mouse hippocampus significantly enhanced seizure susceptibility and severity, whereas overexpression of KCTD13 showed the opposite effect. Mechanistically, GluN1, an obligatory subunit of N-methyl-D-aspartic acid receptors (NMDARs), was identified as a potential substrate protein of KCTD13. Further investigation revealed that KCTD13 facilitates lysine-48-linked polyubiquitination of GluN1 and its degradation through the ubiquitin-proteasome pathway. Besides, the lysine residue 860 of GluN1 is the main ubiquitin site. Importantly, dysregulation of KCTD13 affected membrane expression of glutamate receptors and impaired glutamate synaptic transmission. Systemic administration of the NMDAR inhibitor memantine significantly rescued the epileptic phenotype aggravated by KCTD13 knockdown. In conclusion, our results demonstrated an unrecognized pathway of KCTD13-GluN1 in epilepsy, suggesting KCTD13 as a potential neuroprotective therapeutic target for epilepsy.

GSDMD knockdown exacerbates hippocampal damage and seizure susceptibility by crosstalk between pyroptosis and apoptosis in kainic acid-induced temporal lobe epilepsy.

BACKGROUND: Neuronal loss is a vital pathological feature of temporal lobe epilepsy (TLE). However, the exact mechanism of neuronal loss in TLE is not fully understood. Pyroptosis, a novel form of programmed cell death (PCD), has been considered a contributor to the pathogenesis of TLE. However, recent studies have implicated extensive molecular crosstalk among pyroptosis, apoptosis, and necroptosis in various diseases, and they can be transformed to each other according to different contexts. This study aimed to investigate whether gasdermin D (GSDMD)-mediated pyroptosis is involved in the pathogenesis of TLE and whether crosstalk exists in the process of the modulation of pyroptosis. METHODS: The TLE model was established by intra-amygdala injection of kainic acid. The Racine score and local field potential (LFP) recordings were used to assess seizure severity. Western blotting and immunofluorescence were applied to detect the levels and cellular localization of GSDMD. The neuronal loss and type of neuronal death in the bilateral hippocampus were assessed by Nissl staining and flow cytometry analysis. The underlying crosstalk among pyroptosis, apoptosis, and necroptosis was explored by western blot and verified by VX765. RESULTS: GSDMD was significantly upregulated and mainly expressed within the neurons of the hippocampus in the TLE model. Inhibition of pyroptosis by GSDMD knockdown triggered caspase-3-mediated apoptosis, leading to excess neuronal loss and deterioration of epileptic behaviors. Blocking caspase-1 markedly inhibited caspase-3-mediated apoptosis and improved epileptic behaviors under GSDMD knockdown. CONCLUSIONS: Our results demonstrate that GSDMD-mediated pyroptosis is involved in the pathogenesis of TLE. However, inhibition of GSDMD triggers caspase-1-mediated crosstalk between pyroptosis and apoptosis, which exacerbates neuronal loss and seizure susceptibility. Therefore, the complex crosstalk among different forms of PCD should be considered when a potential molecular target in the single PCD pathway is modulated. On the other hand, along with further studies of molecular crosstalk among the PCD pathways, taking advantage of crosstalk to attenuate neuronal loss may provide new insight for the clinical therapy of TLE.

Response to antiepileptic drugs after unsuccessful epilepsy surgery: A multivariate analysis of 103 patients.

OBJECT: Epilepsy patients may still have seizures after surgery, and there have been few studies on the response to antiepileptic drugs (AEDs) after surgery failure. The purpose of this study was to analyze the response to AEDs after unsuccessful epilepsy surgery. METHODS: Patients who underwent unsuccessful epilepsy surgery between January 1999 and January 2019 were evaluated. Patient demographics, etiology, factors related to surgery and AED use patterns were assessed. RESULTS: After excluding the 5 patients who were lost to follow-up and the 2 patients who died, the records of 103 consecutive patients were analyzed. Ninety patients (87.4 %) had seizure recurrence within one year after surgery, 2 (1.9 %) patients had recurrence from one year to two years after surgery, and 11 (10.7 %) patients had recurrence two or more years after surgery (2-10 years). After surgery failure, the patients tried at least 2 kinds of AEDs with different mechanisms for more than 2 years. The average total number of AEDs used was 5.97, the average number of AEDs used before surgery was 3.21, and the average number of AEDs used after surgery was 4.02. After retreatment with AEDs, 10 patients (9.7 %) were seizure-free, 18 patients' (17.5 %) seizures were alleviated, and 75 patients (72.8 %) had seizures as they did prior to the adjustments. The number of AEDs used before and after surgery and the total number of AEDs were not significantly different among the seizure free group, alleviated seizure group and no change group. There were no significant differences in seizure onset age, surgery age, etiology, time between seizure onset and surgery, magnetic resonance imaging, seizure type, localization and lateralization of the surgery site among the three groups. CONCLUSIONS: The results showed that a small percentage of patients (27.2 %) who undergo unsuccessful epilepsy surgery benefit from AED adjustments; however, the vast majority of patients (72.8 %) do not benefit from AED adjustments.

Inhibition of Nwd1 activity attenuates neuronal hyperexcitability and GluN2B phosphorylation in the hippocampus.

BACKGROUND: NACHT and WD repeat domain-containing protein 1 (Nwd1) is a member of the innate immune protein subfamily. Nwd1 contributes to the androgen receptor signaling pathway and is involved in axonal growth. However, the mechanisms that underlie pathophysiological dysfunction in seizures remain unclear. METHODS: Biochemical methods were used to assess Nwd1 expression and localization in a mouse model of kainic acid (KA)-induced acute seizures and temporal lobe epilepsy (TLE) patients. Electrophysiological recordings were used to measure the role of Nwd1 in regulating synaptic transmission and neuronal hyperexcitability in a model of magnesium-free-induced seizure in vitro. Behavioral experiments were performed, and seizure-induced pathological changes were evaluated in a KA-induced seizure model in vivo. GluN2B expression was measured and its correlation with Tyr1472-GluN2B phosphorylation was analyzed in primary hippocampal neurons. FINDINGS: We demonstrated high protein levels of Nwd1 in brain tissues obtained from mice with acute seizures and TLE patients. Silencing Nwd1 in mice using an adeno-associated virus (AAV) profoundly suppressed neuronal hyperexcitability and the occurrence of acute seizures, which may have been caused by reducing GluN2B-containing NMDA receptor-dependent glutamatergic synaptic transmission. Moreover, the decreased activation of Nwd1 reduced GluN2B expression and the phosphorylation of the GluN2B subunit at Tyr1472. INTERPRETATION: Here, we report a previously unrecognized but important role of Nwd1 in seizure models in vitro and in vivo, i.e., modulating the phosphorylation of the GluN2B subunit at Tyr1472 and regulating neuronal hyperexcitability. Meanwhile, our findings may provide a therapeutic strategy for the treatment of epilepsy or other hyperexcitability-related neurological disorders. FUND: The funders have not participated in the study design, data collection, data analysis, interpretation, or writing of the report.

TMEM25 modulates neuronal excitability and NMDA receptor subunit NR2B degradation.

The expression of the transmembrane protein 25 gene (Tmem25) is strongly influenced by glutamate ionotropic receptor kainate type subunit 4, and its function remains unknown. Here, we showed that TMEM25 was primarily localized to late endosomes in neurons. Electrophysiological experiments suggested that the effects of TMEM25 on neuronal excitability were likely mediated by N-methyl-d-aspartate receptors. TMEM25 affected the expression of the N-methyl-d-aspartate receptor NR2B subunit and interacted with NR2B, and both were colocalized to late endosome compartments. TMEM25 induced acidification changes in lysosome compartments and accelerated the degradation of NR2B. Furthermore, TMEM25 expression was decreased in brain tissues from patients with epilepsy and epileptic mice. TMEM25 overexpression attenuated the behavioral phenotypes of epileptic seizures, whereas TMEM25 downregulation exerted the opposite effect. These results provide some insights into TMEM25 biology in the brain and the functional relationship between TMEM25 and epilepsy.

Age- and Nicotine-Associated Gene Expression Changes in the Hippocampus of APP/PS1 Mice.

The etiology of Alzheimer's disease (AD) has been intensively studied. However, little is known about the molecular alterations in early-stage and late-stage AD. Hence, we performed RNA sequencing and assessed differentially expressed genes (DEGs) in the hippocampus of 18-month and 7-month-old APP/PS1 mice. Moreover, the DEGs induced by treatment with nicotine, the nicotinic acetylcholine receptor agonist that is known to improve cognition in AD, were also analyzed in old and young APP/PS1 mice. When comparing old APP/PS1 mice with their younger littermates, we found an upregulation in genes associated with calcium overload, immune response, cancer, and synaptic function; the transcripts of 14 calcium ion channel subtypes were significantly increased in aged mice. In contrast, the downregulated genes in aged mice were associated with ribosomal components, mitochondrial respiratory chain complex, and metabolism. Through comparison with DEGs in normal aging from previous reports, we found that changes in calcium channel genes remained one of the prominent features in aged APP/PS1 mice. Nicotine treatment also induced changes in gene expression. Indeed, nicotine augmented glycerolipid metabolism, but inhibited PI3K and MAPK signaling in young mice. In contrast, nicotine affected genes associated with cell senescence and death in old mice. Our study suggests a potential network connection between calcium overload and cellular signaling, in which additional nicotinic activation might not be beneficial in late-stage AD.

Transgenic overexpression of furin increases epileptic susceptibility.

The proprotein convertase Furin plays crucial roles in the pathology of many diseases. However, the specific role of furin in epilepsy remains unclear. In our study, furin protein was increased in the temporal neocortex of epileptic patients and in the hippocampus and cortex of epileptic mice. The furin transgenic (TG) mice showed increased susceptibility to epilepsy and heightened epileptic activity compared with wild-type (WT) mice. Conversely, lentivirus-mediated knockdown of furin restrained epileptic activity. Using whole-cell patch clamp, furin knockdown and overexpression influenced neuronal inhibitory by regulating postsynaptic gamma-aminobutyric acid A receptor (GABAAR)-mediated synaptic transmission. Importantly, furin influenced the expression of GABAAR ß2/3 membrane and total protein in epileptic mice by changing transcription level of GABAAR ß2/3, not the protein degradation. These results reveal that furin may regulate GABAAR-mediated inhibitory synaptic transmission by altering the transcription of GABAAR ß2/3 subunits in epilepsy; this finding could provide new insight into epilepsy prevention and treatment.

GPR40 modulates epileptic seizure and NMDA receptor function.

Epilepsy is a common neurological disease, and approximately 30% of patients do not respond adequately to antiepileptic drug treatment. Recent studies suggest that G protein-coupled receptor 40 (GPR40) is expressed in the central nervous system and is involved in the regulation of neurological function. However, the impact of GPR40 on epileptic seizures remains unclear. In this study, we first reported that GPR40 expression was increased in epileptic brains. In the kainic acid-induced epilepsy model, GPR40 activation after status epilepticus alleviated epileptic activity, whereas GPR40 inhibition showed the opposite effect. In the pentylenetetrazole-induced kindling model, susceptibility to epilepsy was reduced with GPR40 activation and increased with GPR40 inhibition. Whole-cell patch-clamp recordings demonstrated that GPR40 affected N-methyl-d-aspartate (NMDA) receptor-mediated synaptic transmission. Moreover, GPR40 regulated NR2A and NR2B expression on the surface of neurons. In addition, endocytosis of NMDA receptors and binding of GPR40 with NR2A and NR2B can be regulated by GPR40. Together, our findings indicate that GPR40 modulates epileptic seizures, providing a novel antiepileptic target.

ZDHHC8 critically regulates seizure susceptibility in epilepsy.

Epilepsy is one of the most prevalent and drug-refractory neurological disorders. Zinc finger DHHC-type containing 8 (ZDHHC8) is a putative palmitoyltransferase that is highly expressed in the brain. However, the impact of ZDHHC8 on seizures remains unclear. We aimed to explore the association of ZDHHC8 with epilepsy and investigate its in epileptogenesis in in vivo and in vitro models through behavioral, electrophysiological, and pathological studies. We used kainic acid- and pilocarpine-induced C57BL/6 mice and magnesium-free-induced pyramidal neurons as experimental epileptic models in this study. We first found increased ZDHHC8 expression in the brains of temporal lobe epilepsy (TLE) patients, similar to that observed in chronic epileptic mice, strongly suggesting that ZDHHC8 is correlated with human epilepsy. In the in vitro seizure models, knocking down ZDHHC8 using recombinant adeno-associated virus (rAAV) delayed seizure precipitation and decreased chronic spontaneous recurrent seizures (SRSs) and epileptiform-like discharges, while ZDHHC8 overexpression had the opposite effect. ZDHHC8 levels were consistent with seizure susceptibility in induced mice with SRSs. In an in vitro magnesium-free model, neuronal hyperexcitability and hypersynchrony were reduced in ZDHHC8-knockdown neurons but were increased in ZDHHC8-overexpressing neurons. To further explore the potential mechanisms, we observed that ZDHHC8 had a significant modulatory effect on 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA) receptor-related excitatory, but not inhibitory, glutamatergic synaptic neurotransmission, further affecting the inward rectification of AMPA currents in acute hippocampal slices in whole-cell recordings. ZDHHC8 facilitated GluA1 trafficking to the neuronal surface in the hippocampus, as shown by immunoprecipitation and Western blotting. These results suggest that ZDHHC8 may promote the generation and propagation of seizures in humans and that knocking down ZDHHC8 might produce anti-epileptogenic effects in drug-resistant epilepsy. Our study provides evidence that may facilitate the development of an alternative approach for the treatment of epilepsy by modulating AMPA/GluA1-mediated neurotransmission.

Inhibition of Cgkii Suppresses Seizure Activity and Hippocampal Excitation by Regulating the Postsynaptic Delivery of Glua1.

BACKGROUND/AIMS: The imbalance between excitation and inhibition is a defining feature of epilepsy. GluA1 is an AMPA receptor subunit that can strengthen excitatory synaptic transmission when upregulated in the postsynaptic membrane, which has been implicated in the pathogenesis of epilepsy. cGKII, a cGMP-dependent protein kinase, regulates the GluA1 levels at the plasma membrane. METHODS: To explore the role of cGKII in epilepsy, we investigated the expression of cGKII in patients with temporal lobe epilepsy (TLE) and in a pilocarpine-induced rat model and then performed behavioral, histological, and electrophysiological analyses by applying either a cGKII agonist or inhibitor in the hippocampus of the animal model. RESULTS: cGKII expression was upregulated in the epileptogenic brain tissues of both humans and rats. Pharmacological activation or inhibition of cGKII induced changes in epileptic behaviors in vivo and epileptic discharges in vitro. Further studies indicated that cGKII activation disrupted the balance of excitation and inhibition due to strengthened AMPAR-mediated excitatory synaptic transmission. Moreover, cGKII regulated epileptic seizures by phosphorylating GluA1 at Ser845 to modulate the expression and function of GluA1 in the postsynaptic membrane. CONCLUSION: These results suggest that cGKII plays a key role in seizure activity and could be a potential therapeutic target for epilepsy.

Furin promotes dendritic morphogenesis and learning and memory in transgenic mice.

Furin is a proprotein convertase implicated in a variety of pathological processes including neurodegenerative diseases. However, the role of furin in neuronal plasticity and learning and memory remains to be elucidated. Here, we report that in brain-specific furin transgenic (Furin-Tg) mice, the dendritic spine density and proliferation of neural progenitor cells were significantly increased. These mice exhibited enhanced long-term potentiation (LTP) and spatial learning and memory performance, without alterations of miniature excitatory/inhibitory postsynaptic currents. In the cortex and hippocampus of Furin-Tg mice, the ratio of mature brain-derived neurotrophic factor (mBDNF) to pro-BDNF, and the activities of extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) were significantly elevated. We also found that hippocampal knockdown of CREB diminished the facilitation of LTP and cognitive function in Furin-Tg mice. Together, our results demonstrate that furin enhances dendritic morphogenesis and learning and memory in transgenic mice, which may be associated with BDNF-ERK-CREB signaling pathway.

POSH participates in epileptogenesis by increasing the surface expression of the NMDA receptor: a promising therapeutic target for epilepsy.

OBJECTIVES: Plenty of SH3 (POSH) was originally found to be a key regulator of neuronal apoptosis, axon outgrowth, and neuronal migration. However, the role of POSH in epilepsy has not been reported. METHODS: We investigated the expression of POSH in patients with intractable temporal epilepsy (TLE) and in a kainic acid (KA)-induced mouse model, and then we performed behavioral, electrophysiological and biochemical analyses after the lentivirus (LV)-mediated knockdown or overexpression of POSH in the KA-induced model. RESULTS: POSH overexpression shortened the latency of seizure onset, increased the frequency of spontaneous recurrent seizures, and increased the frequency of electrical epileptic discharges, while POSH knockdown had contrasting effects. Whole-cell patch-clamp recordings confirmed that POSH overexpression and knockdown were associated with increased and decreased miniature excitatory postsynaptic currents (mEPSCs) and N-methyl-D-aspartate receptor (NMDAR)-mediated currents, respectively. Finally, co-immunoprecipitation showed that POSH and NMDA receptor subunit 1 (NMDAR1) precipitated with each other, and western blot analysis revealed that the surface expression of NMDAR1 was altered in the hippocampus of epileptic mice. CONCLUSION: These results show that POSH plays a critical role in the progression of epileptic seizures via NMDAR trafficking and suggest that the protein is a novel target for the treatment of human epilepsy.

Tetrachlorobenzoquinone triggers the cleavage of Bid and promotes the cross-talk of extrinsic and intrinsic apoptotic signalings in pheochromocytoma (PC) 12 cells.

Although there are few studies suggested PCP exposure induced developmental and behavioral disorders, however, the occurrence of neurotoxicity and PCP has not been firmly established. Tetrachlorobenzoquinone (TCBQ) is a reactive metabolite of environmental pollutant pentachlorophenol (PCP). To the best of our knowledge, there has no information regarding to the neurological toxic effect of TCBQ available. Here, we demonstrated that TCBQ induces cytotoxicity in pheochromocytoma PC12 cell line, and the mode-of-action analysis indicated the involvement of apoptotic signalings, such as the activation of caspase family proteins, the increased expressions of Fas and Fas-associated death domain (FADD), the loss of mitochondrial membrane potential (MMP), the release of cytochrome c (Cyt c) and the cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP). BI-6C9, a specific BH3-interacting domain death agonist (Bid) inhibitor, repressed TCBQ-induced Bid truncation, along with the activation of caspase 3 and the release of Cyt c, suggested the cross-talk of extrinsic and intrinsic apoptotic signalings. Furthermore, the inhibition of caspase 8 impaired TCBQ-induced the activation of caspase 3, as well as the release of Cyt c and the cleavage of Bid, suggesting caspase 8 acting as the upstream molecule of Bid, and TCBQ-induced apoptosis is initiated via caspase 8, leads to the activation of caspase 9/3 through Bid-mediated amplification loop. Finally, the pretreatment of antioxidant NAC ameliorated Fas, FADD and caspase 8/3 expressions, which illustrated that TCBQ-induced apoptotic signaling is ROS dependent. Taken together, these results indicated that the cleavage of Bid may play an important role in TCBQ-induced neurotoxicity which promotes the cross-talk of extrinsic and intrinsic apoptotic signalings in PC12 cells.

Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response, and calcium release.

Organisms are able to respond to environmental insult to maintain cellular homeostasis, which include the activation of a wide range of cellular adaptive responses with tightly controlled mechanisms. The endoplasmic reticulum (ER) is an organelle responsible for protein folding and calcium storage. ER stress leads to the accumulation of unfolded proteins in the ER lumen. To be against or respond to this effect, cells have a comprehensive signaling system, called unfolded protein response (UPR), to restore homeostasis and normal ER function or activate the cell death program. Therefore, it is critical to understand how environmental insult regulates the ingredients of ER stress and UPR signalings. Previously, we have demonstrated that polychlorinated biphenyl (PCB) quinone caused oxidative stress, cytotoxicity, genotoxicity, and apoptosis in HepG2 cells. Here, we investigated the role of a PCB quinone, PCB29-pQ on ER stress, UPR, and calcium release. PCB29-pQ markedly increased the hallmark genes of ER stress, namely, glucose-regulated protein 78 (GRP78), GRP94, and C/EBP homologous protein (CHOP) on both protein and mRNA levels in HepG2 cells. We also confirmed PCB29-pQ induced ER morphological defects by using transmission electron microscopy. Moreover, PCB29-pQ induced intracellular calcium accumulation and calpain activity, which were significantly inhibited by the pretreatment of BAPTA-AM (Ca(2+) chelator). These results were correlated with the outcome that PCB29-pQ induces ER stress-related apoptosis through caspase family gene 12, while salubrinal and Z-ATAD-FMK (a specific inhibitor of caspase 12) partially ameliorated this effect, respectively. N-Acetyl-l-cysteine (NAC) scavenged ROS formation and consequently alleviated PCB29-pQ-induced expression of ER stress-related genes. In conclusion, our result demonstrated for the first time that PCB quinone leads to ROS-dependent induction of ER stress, and UPR and calcium release in HepG2 cells, and the evaluation of the perturbations of ER stress, UPR, and calcium signaling provide further information on the mechanisms of PCB-induced toxicity.

Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis.

Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis.

Tetrachlorobenzoquinone activates Nrf2 signaling by Keap1 cross-linking and ubiquitin translocation but not Keap1-Cullin3 complex dissociation.

Tetrachlorobenzoquinone (TCBQ), a metabolite of industrial herbicide pentachlorophenol, showed hepatotoxicity and genotoxicity through reactive oxygen species (ROS) mechanism in vivo and in vitro models. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a cellular sensor of oxidative or electrophilic stress, which controls the expression of detoxifying enzymes and antioxidant proteins. Using the human hepatoma HepG2 cell line as an in vitro model, we demonstrated a significant induction of Nrf2 but not its negative regulator Kelch-like ECH-associated protein 1 (Keap1), following exposure to TCBQ. Also, our results clearly demonstrated the translocation of cytosolic Nrf2 into the nucleus. After translocation, Nrf2 subsequently binds to the antioxidant response element (ARE), up-regulated heme oxygenase-1 (HO-1), and NADH quinone oxidoreductase subunit 1 (NQO1), which may be considered as an antioxidative response to TCBQ-intoxication. The luciferase reporter assay confirmed the formation of the Nrf2-ARE complex. Furthermore, mechanism studies proposed that TCBQ promoted the formation of the Keap1 cross-linking dimer, a ubiquitination switch from Nrf2 to Keap1 but not the dissociation of the Keap1-Cullin3 (Cul3) complex.

Polychlorinated biphenyl quinone induces mitochondrial-mediated and caspase-dependent apoptosis in HepG2 cells.

Polychlorinated biphenyl (PCB) quinones are known to cause toxic effects, but their mechanisms are quite unclear. In this study, we examined whether 2,3,5-trichloro-6-phenyl-[1,4]benzoquinone, PCB29-pQ, induces cell death via apoptosis pathway. Our result showed PCB29-pQ exposure decreased HepG2 cell viability in a time-dependent manner. Lactate dehydrogenase leakage assay also implied the cytotoxicity of PCB29-pQ. 4',6-Diamidino-2-phenylindole dihydrochloride staining and flow cytometry assays both confirmed PCB29-pQ caused dose-dependent apoptotic cell death in HepG2 cells. Furthermore, we found that PCB29-pQ exposure increased cellular reactive oxygen species (ROS) level, decreased mitochondrial membrane potential and induced the translocation of cytochrome c from mitochondria into cytosol in HepG2 cells. Moreover, PCB29-pQ exposure induced B-cell lymphoma 2 (Bcl-2) downregulation and Bcl-2-associated X (Bax) upregulation, poly(ADP-ribose) polymerase cleavage, accompanied with the increased caspase-3/9 and p53 expressions. Taking together, these results suggested PCB29-pQ induced HepG2 cells apoptosis through a ROS-driven, mitochondrial-mediated and caspase-dependent pathway.

Tetrachloro-p-benzoquinone induces hepatic oxidative damage and inflammatory response, but not apoptosis in mouse: the prevention of curcumin.

This study investigated the protective effects of curcumin on tetrachloro-p-benzoquinone (TCBQ)-induced hepatotoxicity in mice. TCBQ-treatment causes significant liver injury (the elevation of serum AST and ALT activities, histopathological changes in liver section including centrilobular necrosis and inflammatory cells), oxidative stress (the elevation of TBAR level and the inhibition of SOD and catalase activities) and inflammation (up-regulation of iNOS, COX-2, IL-1ß, IL-6, TNF-α and NF-κB). However, these changes were alleviated upon pretreatment with curcumin. Interestingly, TCBQ has no effect on caspase family genes or B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X (Bax) protein expressions, which implied that TCBQ-induced hepatotoxicity is independent of apoptosis. Moreover, curcumin was shown to induce phase II detoxifying/antioxidant enzymes HO-1 and NQO1 through the activation of nuclear factor erythroid-derived 2-like 2 (Nrf2). In summary, the protective mechanisms of curcumin against TCBQ-induced hepatoxicity may be related to the attenuation of oxidative stress, along with the inhibition of inflammatory response via the activation of Nrf2 signaling.