ALKBH5 targets ACSL4 mRNA stability to modulate ferroptosis in hyperbilirubinemia-induced brain damage.

Bilirubin-induced brain damage is a serious clinical consequence of hyperbilirubinemia, yet the underlying molecular mechanisms remain largely unknown. Ferroptosis, an iron-dependent cell death, is characterized by iron overload and lipid peroxidation. Here, we report a novel regulatory mechanism of demethylase AlkB homolog 5 (ALKBH5) in acyl-coenzyme A synthetase long-chain family member 4 (ACSL4)-mediated ferroptosis in hyperbilirubinemia. Hyperdifferential PC12 cells and newborn Sprague-Dawley rats were used to establish in vitro and in vivo hyperbilirubinemia models, respectively. Proteomics, coupled with bioinformatics analysis, first suggested the important role of ferroptosis in hyperbilirubinemia-induced brain damage. In vitro experiments showed that ferroptosis is activated in hyperbilirubinemia, and ferroptosis inhibitors (desferrioxamine and ferrostatin-1) treatment effectively alleviates hyperbilirubinemia-induced oxidative damage. Notably, we observed that the ferroptosis in hyperbilirubinemia was regulated by m6A modification through the downregulation of ALKBH5 expression. MeRIP-seq and RIP-seq showed that ALKBH5 may trigger hyperbilirubinemia ferroptosis by stabilizing ACSL4 mRNA via m6A modification. Further, hyperbilirubinemia-induced oxidative damage was alleviated through ACSL4 genetic knockdown or rosiglitazone-mediated chemical repression but was exacerbated by ACSL4 overexpression. Mechanistically, ALKBH5 promotes ACSL4 mRNA stability and ferroptosis by combining the 669 and 2015 m6A modified sites within 3' UTR of ACSL4 mRNA. Our findings unveil a novel molecular mechanism of ferroptosis and suggest that m6A-dependent ferroptosis could be an underlying clinical target for the therapy of hyperbilirubinemia.

ROS/mtROS promotes TNTs formation via the PI3K/AKT/mTOR pathway to protect against mitochondrial damages in glial cells induced by engineered nanomaterials.

BACKGROUND: As the demand and application of engineered nanomaterials have increased, their potential toxicity to the central nervous system has drawn increasing attention. Tunneling nanotubes (TNTs) are novel cell-cell communication that plays a crucial role in pathology and physiology. However, the relationship between TNTs and nanomaterials neurotoxicity remains unclear. Here, three types of commonly used engineered nanomaterials, namely cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2NPs), and multi-walled carbon nanotubes (MWCNTs), were selected to address this limitation. RESULTS: After the complete characterization of the nanomaterials, the induction of TNTs formation with all of the nanomaterials was observed using high-content screening system and confocal microscopy in both primary astrocytes and U251 cells. It was further revealed that TNT formation protected against nanomaterial-induced neurotoxicity due to cell apoptosis and disrupted ATP production. We then determined the mechanism underlying the protective role of TNTs. Since oxidative stress is a common mechanism in nanotoxicity, we first observed a significant increase in total and mitochondrial reactive oxygen species (namely ROS, mtROS), causing mitochondrial damage. Moreover, pretreatment of U251 cells with either the ROS scavenger N-acetylcysteine or the mtROS scavenger mitoquinone attenuated nanomaterial-induced neurotoxicity and TNTs generation, suggesting a central role of ROS in nanomaterials-induced TNTs formation. Furthermore, a vigorous downstream pathway of ROS, the PI3K/AKT/mTOR pathway, was found to be actively involved in nanomaterials-promoted TNTs development, which was abolished by LY294002, Perifosine and Rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively. Finally, western blot analysis demonstrated that ROS and mtROS scavengers suppressed the PI3K/AKT/mTOR pathway, which abrogated TNTs formation. CONCLUSION: Despite their biophysical properties, various types of nanomaterials promote TNTs formation and mitochondrial transfer, preventing cell apoptosis and disrupting ATP production induced by nanomaterials. ROS/mtROS and the activation of the downstream PI3K/AKT/mTOR pathway are common mechanisms to regulate TNTs formation and mitochondrial transfer. Our study reveals that engineered nanomaterials share the same molecular mechanism of TNTs formation and intercellular mitochondrial transfer, and the proposed adverse outcome pathway contributes to a better understanding of the intercellular protection mechanism against nanomaterials-induced neurotoxicity.

Targeting Mitochondrial Dysfunction With LncRNAs in a Wistar Rat Model of Chronic Obstructive Pulmonary Disease.

BACKGROUND/AIM: Chronic obstructive pulmonary disease (COPD) has become a prominent healthcare issue in recent years. Cigarette smoking (CS) and fine particulate matter (PM2.5) are important causative factors for COPD. This study assessed the aberrant lncRNA profiles in the tissue of rats with COPD caused by CS or PM2.5 Materials and Methods: A COPD rat model was developed using CS (CSM) or PM2.5 (PMM), and lung tissue RNA was extracted. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) were used to investigate the correlations between the distinct lncRNAs and mRNA pathways. A coding-non-coding gene co-expression network (CNC) was constructed by establishing connections between differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) associated with mitochondrial dysfunction and the inflammatory response. RESULTS: A quantitative real-time reverse transcription PCR (qRT-PCR) experiment was performed to verify the expression of the particular lncRNAs. Microarray analysis of lung tissue from the COPD model revealed that 123 and 444 lncRNAs were substantially raised and reduced in PMM vs. the control group (Ctrl), respectively, as were 621 and 1,178 mRNAs. Meanwhile, 81 and 340 lncRNAs were consistently raised and lowered in CSM vs. Ctrl, respectively, as were 408 and 931 mRNAs. GO enrichment and KEGG pathway analysis indicated that the COPD model was connected to inflammatory responses, mitochondrial dysfunction, and others. CONCLUSION: XR_340674, ENSRNOT00000089642, XR_597045, and XR_340651 were decreased, and XR_592469 was elevated. These lncRNAs were shown to be related to mitochondrial dysfunction in the lung tissue of animals exposed to CS or PM2.5.

Microglia-derived exosomal circZNRF1 alleviates paraquat-induced neuronal cell damage via miR-17-5p.

Paraquat (PQ) is an environmental poison that causes clinical symptoms similar to those of Parkinson's disease (PD) in vitro and in rodents. It can lead to the activation of microglia and apoptosis of dopaminergic neurons. However, the exact role and mechanism of microglial activation in PQ-induced neuronal degeneration remain unknown. Here, we isolated the microglia-derived exosomes exposed with 0 and 40 µM PQ, which were subsequently co-incubated with PQ-exposed neuronal cells to simulate intercellular communication. First, we found that exosomes released from microglia caused a change in neuronal cell vitality and reversed PQ-induced neuronal apoptosis. RNA sequencing data showed that these activated microglia-derived exosomes carried large amounts of circZNRF1. Moreover, a bioinformatics method was used to study the underlying mechanism of circZNRF1 in regulating PD, and miR-17-5p was predicted to be its target. Second, an increased Bcl2/Bax ratio could play an anti-apoptotic role. Bcl2 was predicted to be a downstream target of miR-17-5p. Our results showed that circZNRF1 plays an anti-apoptotic role by absorbing miR-17-5p and regulating the binding of Bcl2 after exosomes are internalized by dopaminergic neurons. In conclusion, we demonstrated a new intercellular communication mechanism between microglia and neurons, in which circZNRF1 plays a key role in protecting against PQ-induced neuronal apoptosis through miR-17-5p to regulate the biological process of PD. These findings may offer a novel approach to preventing and treating PD.

Evaluation of neurotoxicity and the role of oxidative stress of cobalt nanoparticles, titanium dioxide nanoparticles, and multiwall carbon nanotubes in Caenorhabditis elegans.

The widespread use of nanomaterials in daily life has led to increased concern about their potential neurotoxicity. Therefore, it is particularly important to establish a simple and reproducible assessment system. Representative nanomaterials, including cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2-NPs), and multiwall carbon nanotubes (MWCNTs), were compared in terms of their neurotoxicity and underlying mechanisms. In 0, 25, 50, and 75 µg/ml of these nanomaterials, the survival, locomotion behaviors, acetylcholinesterase (AchE) activity, reactive oxygen species production, and glutathione-S transferase 4 (Gst-4) activation in wildtype and transgenic Caenorhabditis elegans (C. elegans) were evaluated. All nanomaterials induced an imbalance in oxidative stress, decreased the ratio of survival, impaired locomotion behaviors, as well as reduced the activity of AchE in C. elegans. Interestingly, CoNPs and MWCNTs activated Gst-4, but not TiO2-NPs. The reactive oxygen species scavenger, N-acetyl-l-cysteine, alleviated oxidative stress and Gst-4 upregulation upon exposure to CoNPs and MWCNTs, and rescued the locomotion behaviors. MWCNTs caused the most severe damage, followed by CoNPs and TiO2-NPs. Furthermore, oxidative stress and subsequent activation of Gst-4 were involved in nanomaterials-induced neurotoxicity. Our study provides a comprehensive comparison of the neurotoxicity and mechanisms of typical nanomaterials, which could serve as a model for hazard assessment of environmental pollutants using C. elegans as an experimental model system.

The interplay between lncRNA NR_030777 and SF3B3 in neuronal damage caused by paraquat.

Paraquat (PQ) has been widely acknowledged as an environmental risk factor for Parkinson's disease (PD). However, the interaction between splicing factor and long non-coding RNA (lncRNA) in the process of PQ-induced PD has rarely been studied. Based on previous research, this study focused on splicing factor 3 subunit 3 (SF3B3) and lncRNA NR_030777. After changing the target gene expression level by lentiviral transfection technology, the related gene expression was detected by western blot and qRT-PCR. The expression of SF3B3 protein was reduced in Neuro-2a cells after PQ exposure, and the reactive oxygen species (ROS) scavenger N-acetylcysteine prevented this decline. Knockdown of SF3B3 reduced the PQ-triggered NR_030777 expression increase, and overexpression of NR_030777 reduced the transcriptional and translational level of Sf3b3. Then, knockdown of SF3B3 exacerbated the PQ-induced decrease in cell viability and aggravated the reduction of tyrosine hydroxylase (TH) protein expression. Overexpressing SF3B3 reversed the reduction of TH expression caused by PQ. Moreover, after intervention with the autophagy inhibitor Bafilomycin A1, LC3B-II protein expression was further increased in Neuro-2a cells with the knockdown of SF3B3, indicating that autophagy was enhanced. In conclusion, PQ modulated the interplay between NR_030777 and SF3B3 through ROS production, thereby impairing autophagic flux and causing neuronal damage.

Involvement of NEAT1/PINK1-mediated mitophagy in chronic obstructive pulmonary disease induced by cigarette smoke or PM2.5.

Background: This study sought to explore the underlying mechanism of long non-coding ribonucleic acid nuclear enriched abundant transcript 1 (NEAT1) and PTEN-induced kinase 1 (PINK1)-mediated mitophagy in chronic obstructive pulmonary disease (COPD) induced by cigarette smoke (CS) or fine particular matter (PM2.5). Methods: In total, 30 male Wistar Rats were divided into the following 3 groups: (I) the COPD group exposed to CS (CSM); (II) the COPD group exposed to PM2.5 (PMM); and (III) the control (Ctrl) group. Pulmonary function, the enzyme-linked immunoassay analysis results, the histopathology results, and the ultrastructures of the lung tissues were examined in the 3 groups, and NEAT1 expression levels and the mitophagy-related protein PINK1, Parkin, LC3B, and p62 levels were assessed by quantitative reverse transcription PCR (RT-qPCR) and Western blotting. The A549 cells were transfected with small interfering ribonucleic acid (siRNA) targeting NEAT1, and subsequently stimulated with CS extract (CSE) and PM2.5 suspension (PMS). Mitochondrial dysfunction and enhanced mitophagy were observed, and the expression of the NEAT1/PINK1 pathway was assessed by RT-qPCR and Western blotting. Results: Both the CSM and PMM groups had a lower tidal volume (VT), minute ventilation (MV), and a higher respiratory rate (f) than the Ctrl group. The interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha levels in the serum and bronchoalveolar lavage fluid of the CSM and PMM groups were significantly increased. The histological examination results revealed airway remodeling, the formation of pulmonary bullae, and emphysema in the CSM and PMM groups. Subsequently, the ultrastructures of the lung tissues in the CSM and PMM groups showed mitochondrial swelling and autophagosomes. Additionally, NEAT1 expression, the level of the mitophagy-related protein PINK1, Parkin, and the ratio of LC3-II/I increased synchronously. Further, NEAT1 siRNA blocked PINK1 expression, inhibited mitochondrial dysfunctions, and mitophagy activation in the A549 cells exposed to CSE or PMS. Conclusions: Our results suggest that CS and PM2.5 exposure induce mitochondrial dysfunction, and the NEAT1/PINK1 pathway plays a critical role in the occurrence and development of COPD by regulating mitophagy.

Paraquat-induced oxidative stress regulates N6-methyladenosine (m6A) modification of long noncoding RNAs in Neuro-2a cells.

Paraquat (PQ) is a ubiquitously applied herbicide. Long-term PQ exposure with low dose has been reported to induce abnormal expression of long non-coding RNAs (lncRNAs) in brain nerve cells, which could further lead to Parkinson's disease (PD). N6-methyladenosine (m6A) modification has recently been identified as having an important role in regulating the function of lncRNAs. However, how m6A modification regulates lncRNAs following PQ exposure remains largely unknown. Herein, this study reported m6A modification of lncRNAs in mouse neuroblastoma cells (Neuro-2a) following PQ induced reactive oxide species (ROS). M6A sequencing was performed to explore the m6A modificated pattern of lncRNAs in Neuro-2a cells which were treated with 200 µM PQ for 3 h. It was found that PQ hypermethylated total RNA and changed the expression of m6A methyltransferase and demethylase proteins, which leading to the alteration of m6A modification of lncRNAs. Furthermore, the functional analysis further revealed that N-acetyl-L-cysteine (NAC)，a ROS scavengers, partly reversed PQ-induced distinct m6A modificated pattern of lncRNAs. In addition, tow specific m6A modified lncRNAs were identified: cell division cycle 5-like (lncRNA CDC5L) and signal transducer and activator of transcription 3 (lncRNA STAT3), which could influence downstream autophagy related biological function. In summary, this work could potentially contribute to the new insight of lncRNAs m6A modification mechanism in the field of environmental toxicology.

N6-methyladenosine(m6A) demethylase FTO regulates cellular apoptosis following cobalt-induced oxidative stress.

Cobalt is an environmental toxicant that is known to damage human health. However, the molecular mechanisms underlying cobalt-induced neurotoxicity have not been elucidated in detail. In the present research, we used human neuroglioma H4 cells as an in vitro model. Cells were exposed to CoCl2 (0, 100, 200, 400 µM) for 24 h. We performed m6A sequencing techniques and constructed FTO-knockdown/FTO-overexpressing cells to investigate the role of FTO-mediated m6A modification in regulating apoptosis following CoCl2 induced oxidative stress. Our study has shown CoCl2 exposure led to the decrease of demethylase FTO as well as elevated oxidative stress. However, NAC treatment could partly reverse the reduction of FTO expression as well as the degree of ROS via eliminating oxidative stress. Meanwhile, MeRIP-seq and RNA-seq further revealed the potential function m6A modification in regulating apoptosis. More importantly, KEGG pathway and Gene ontology (GO) analyses further elucidated that the differentially m6A-modified genes were aggregated in apoptosis-related pathways. Mechanistic analysis indicated that knockdown of FTO facilitated CoCl2-induced apoptosis via caspase activation and G1/S cell cycle arrest. Nevertheless, overexpression of FTO partly attenuated the increased apoptosis following CoCl2 exposure. More notably, we observed that FTO regulated apoptosis in an m6A-dependent manner. Therefore, our findings reveal that CoCl2 induced ROS affected the m6A modification of apoptosis-related genes by decreasing the expression of FTO, thereby resulting in the activation of apoptosis. These findings provide important insights into CoCl2-induced apoptosis and m6A modification and propose a novel strategy for studying environmental toxicant-related neurodegeneration.

Paraquat-induced oxidative stress regulates N6-methyladenosine (m6A) modification of circular RNAs.

Paraquat (PQ), a widely used herbicide and well-known oxidative stress inducer, has been linked to numerous neurodegenerative diseases, but the underlying mechanism(s) remains unknown. Circular RNAs (circRNAs) have recently been reported to be associated with oxidative stress in Parkinson's disease. Herein, we performed methylated RNA immunoprecipitation and RNA sequencing assays for mouse neuroblastoma (Neuro-2a) cells and successfully established a positive link between the alteration of circRNAs driven by m6A modification and PQ-induced oxidative stress. We observed oxidative stress and antioxidative stress present distinct m6A modification pattern of circRNAs as well as biological effect. Gene ontology and pathway analysis predicted that differentially m6A-methylated and expressed circRNAs are highly clustered in pathways associated with function and development of nervous system, including axon cargo transport, nervous system development, long-term potentiation, and neurotrophic signaling pathways. Moreover, we demonstrated that the alteration of m6A-methylated circRNAs upon PQ exposure could be partially reversed by N-acetylcysteine pretreatment. The mechanistic analysis further demonstrated that N-acetylcysteine pretreatment attenuated the decreased expression of target genes (UBC and PPP2CA) induced by PQ. These findings revealed distinct patterns of differentially m6A-modified circRNAs, indicating that m6A could participate in a specific regulatory network of circRNAs to modulate the expression of downstream genes in response to PQ-induced oxidative stress. In conclusion, our work established a link between m6A modification of circRNAs and PQ-induced oxidative stress, and further studies are required to explore the underlying molecular mechanisms associated with PQ-induced neurotoxicity.

The negative role of histone acetylation in cobalt chloride-induced neurodegenerative damages in SHSY5Y cells.

Cobalt has been known for its neurotoxicity in numerous studies. However, the molecular mechanism underlying cobalt-induced neurotoxicity remains largely unknown. In this study, two neuroblastoma (SHSY5Y and N2a) cell lines and a phaeochromocytoma (PC12) line were used as in vitro models. Cells were treated for 24 h with 50, 100, 200, 300, 400 µM cobalt chloride (CoCl2) or cultured with 300 µM CoCl2 for 4, 8, 12 and 24 h to investigate the effects of histone acetylation on CoCl2-induced neurodegenerative damages. Our findings demonstrate that CoCl2 suppresses the acetylation of histone H3 and H4 in a time-dependent and dosage-dependent manner. Furthermore, CoCl2 selectively decreases the expression and activity of histone acetyltransferase (HAT) but has no effects on histone deacetylase (HDAC) in SHSY5Y cells. More importantly, we show that 100 ng/mL HDAC inhibitor trichostatin (TSA) pre-treatment partly attenuates 300 µM CoCl2-induced neurodegenerative damages in SHSY5Y cells. Mechanistic analyses show that CoCl2-induced neurodegenerative damages are associated with the dysfunction of APP, BACE1, PSEN1, NEP and HIF-1α genes, whose expression are partly mediated by histone modification. In summary, we demonstrate that histone acetylation is involved in CoCl2-induced neurodegenerative damages. Our study indicates an important connection between histone modification and the pathological process of neurodegenerative damages and provides a mechanism for cobalt-mediated epigenetic regulation.

Global N6-methyladenosine profiling of cobalt-exposed cortex and human neuroblastoma H4 cells presents epitranscriptomics alterations in neurodegenerative disease-associated genes.

Excessive exposure to cobalt (Co) is known to make adverse impact on the nervous system, but its detailed mechanisms of neurotoxicity have yet to be determined. In this study, C57BL/6 mice (0, 4, 8, 16 mg/kg CoCl2, 30 days) and human neuroblastoma H4 cells (0, 100, 400, 600 µM CoCl2) were used as in vivo and in vitro models. Our results revealed that CoCl2 intraperitoneal injection caused significant impairments in learning and memory, as well as pathological damage in the nervous system. We further certificated the alteration of m6A methylation induced by CoCl2 exposure. Our findings demonstrate for the first time, significant differences in the degree of m6A modification, the biological function of m6A-modified transcripts between cortex and H4 cell samples. Specifically, MeRIP-seq and RNA-seq elucidate that CoCl2 exposure results in differentially m6A-modified and expressed genes, which were enriched in pathways involving synaptic transmission, and central nervous system (CNS) development. Mechanistic analyses revealed that CoCl2 remarkably changed m6A modification level by affecting the expression of m6A methyltransferase and demethylase, and decreasing the activity of demethylase. We observed variation of m6A modification in neurodegenerative disease-associated genes upon CoCl2 exposure and identified regulatory strategy between m6A and potential targets mRNA. Our novel findings provide novel insight into the functional roles of m6A modification in neurodegenerative damage caused by environmental neurotoxicants and identify Co-mediated specific RNA regulatory strategy for broadening the epigenetic regulatory mechanism of RNA induced by heavy metals.

Reactive oxygen species regulate miR-17-5p expression via DNA methylation in paraquat-induced nerve cell damage.

There is emerging evidence suggesting that oxidative stress and DNA methylation can alter miRNA expression. However, little is known on the mechanism of miR-17-5p expression changes in paraquat (PQ)-induced nerve cell damage. In the present study, neuro-2a cells were pretreated with antioxidant N-acetylcysteine (NAC) or DNA methylation inhibitor decitabine (DAC), then exposed to different concentrations of PQ, while the expression levels of miR-17-5p were detected by qRT-PCR. Here, it is showed that PQ downregulated the expression of miR-17-5p dose-dependently in neuro-2a cells. The DNA methylation level was upregulated after PQ exposure, while downregulated with the pretreatment of NAC in the above content, detected by 5-mC immunofluorescence technique. The interaction effect of NAC and PQ in alternating DNA methylation level was further confirmed by flow cytometry. NAC and DAC individually had an interaction effect in PQ-induced nerve cell damage. After using NAC, PQ-induced ROS elevation and DNA methylation are reduced, thereby preventing the proapoptotic effect of miR-17-5p. Above all, PQ can induce DNA methylation variations through ROS production, leading to the downregulation of miR-17-5p expression in PQ-induced nerve cell damage.

Role of histone acetylation in activation of nuclear factor erythroid 2-related factor 2/heme oxygenase 1 pathway by manganese chloride.

Manganese neurotoxicity is characterized by Parkinson-like symptoms with degeneration of dopaminergic neurons in the basal ganglia as the principal pathological feature. Manganese neurotoxicity studies may contribute to a good understanding of the mechanism of Parkinson's disease (PD). In this study, we first confirmed that MnCl2 can promote the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) protein in the nucleus or cytoplasm while increasing the binding activity of Nrf2 and antioxidant response elements, further promoting the expression of downstream target gene heme oxygenase 1 (HO-1) and leading to increase levels of reactive oxygen species (ROS) and reduce the levels of reduced glutathione (GSH). Second, we investigated the role of histone acetylation in the activation of Nrf2/HO-1 pathway by manganese chloride in rat adrenal pheochromocytoma (PC12) cells. Histone acetyltransferase inhibitor (anacardic acid) and histone deacetylase inhibitor (trichostatin A, TSA) were used as pretreatment reagents to adjust the level of histone acetylation. Here, we show that downregulation of histone acetylation can inhibit Mn-induced Nrf2 nuclear translocation and further inhibits the Mn-activated Nrf2/HO-1 pathway. This downregulation also promotes manganese-induced increase of ROS and decrease of GSH in neurons. These results suggest that the downregulation of histone acetylation may play an important role in the neurotoxicity caused by manganese and that TSA may provide new ideas and targets in treating manganese-induced Parkinson's syndrome and PD.