ALKBH5-mediated N6-methyladenosine modification of HO-1 mRNA regulates ferroptosis in cobalt-induced neurodegenerative damage.

The utilization of Cobalt (Co) has surged due to it is critical role in renewable energy technologies and other high-tech applications. Concurrently, the potential health risks associated with Co exposure have raised concerns. Previous studies, including our own, have shown that Co can impair learn and memory functions as an epigenetic hazard, even at low concentrations. In this study, we explore the mechanisms of Co-induced ferroptosis in neurodegenerative damage both in vivo and in vitro, focusing on the epigenetic regulation by N6-methyladenosine (m6A) demethylase alkB homolog 5 (ALKBH5). We identify heme oxygenase-1 (HO-1) as a direct target gene of ALKBH5, playing a crucial role in mitigating Co-induced ferroptosis. ALKBH5 deficiency affects the post-transcriptional regulation of HO-1 through m6A modification, which in turn influences mRNA's stability, intracellular distribution, and alternative splicing, thereby enhancing susceptibility to Co-induced ferroptosis. Additionally, we discuss the potential involvement of heterogeneous nuclear ribonucleoprotein M (hnRNPM) in regulating alternative splicing of HO-1 mRNA, potentially mediated by m6A modifications. This study provides new epigenetic insights into the post-transcriptional regulatory mechanisms involved in Co-induced ferroptosis and highlights the broader implications of environmental hazards in neurodegenerative damage.

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.

VGluT2 neuron subtypes in the paraventricular thalamic nucleus regulate depression in paraquat-induced Parkinson's disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disease and approximately one third of patients with PD are estimated to experience depression. Paraquat (PQ) is the most widely used herbicide worldwide and PQ exposure is reported to induce PD with depression. However, the specific brain region and neural networks underlying the etiology of depression in PD, especially in the PQ-induced model, have not yet been elucidated. Here, we report that the VGluT2-positive glutamatergic neurons in the paraventricular thalamic nucleus (PVT) promote depression in the PQ-induced PD mouse model. Our results show that PVTVGluT2 neurons are activated by PQ and their activation increases the susceptibility to depression in PD mice. Conversely, inhibition of PVTVGluT2 neurons reversed the depressive-behavioral changes induced by PQ. Similar to the effects of intervention the soma of PVTVGluT2 neurons, stimulation of their projections into the central amygdaloid nucleus (CeA) also strongly influenced depression in PD mice. PQ induced malfunctioning of the glutamate system and changes in the dendritic and synaptic morphology in the CeA through its role on PVTVGluT2 neuronal activation. In summary, our results demonstrate that PVTVGluT2 neurons are key neuronal subtypes for depression in PQ-induced PD and promote depression processes through the PVTVGluT2-CeA pathway.

Microglial exosomes in paraquat-induced Parkinson's disease: Neuroprotection and biomarker clues.

The exact mechanisms underlying the initiation and exacerbation of Parkinson's disease (PD) by paraquat remain unclear. We have revealed that exosomes mediate neurotoxicity induced by low dose paraquat exposure by transmitting intercellular signaling. Exposure to 40 µM paraquat promoted exosome release from mouse microglia cells (BV2) in vitro. Paraquat exposure at 100 µM caused degeneration of mouse dopaminergic MN9D cells and inhibited microglia exosome uptake by fluorescently labeling exosomes. We established an incubation model for exosomes and dopaminergic neuron cells under PQ treatment. The results indicated that microglial exosomes alleviated degeneration, increasing proliferation and PD-related protein expression of dopaminergic neurons; however, paraquat reversed this effect. Then, through exosome high-throughput sequencing and qRT-PCR experiments, miR-92a-3p and miR-24-3p were observed to transfer from exosomes to dopaminergic neurons, inhibited by paraquat. The specificity of miR-92a-3p and miR-24-3p was verified in PD patients exosomes, indicating the potential diagnostic value of the exosomal miRNAs in paraquat-induced PD. These results suggest glia-neuron communication in paraquat-induced neurodegeneration and may identify stable paraquat-mediated PD biomarkers, offering clues for early recognition and prevention of pesticide-induced degenerative diseases.

LncRNA NR_030777 promotes mitophagy by targeting CDK1-related mitochondrial fission and ATG12 to attenuate paraquat-induced Parkinson's disease.

With the evidence emerging that abnormal expression of long noncoding RNAs (lncRNAs) are involved in onset of Parkinson's disease (PD), the role of NR_030777 contributing to this disease is of great interest. We recently found that a novel lncRNA "NR_030777" demonstrates protective effects on PQ-induced neurodegeneration. However, the underlying molecular mechanisms of NR_030777 in the regulation of mitochondrial fission and mitophagy involved in PQ-induced neuronal damage remain to be explored. NR_030777 brain conditional overexpressing mice as well as in vitro primary neuronal cells from cerebral cortex and Neuro2a cells were adopted. Immunofluorescence, Immunohistochemistry, qRT-PCR and Western blotting were used to evaluate the expression levels of RNA and proteins. RNA immunoprecipitation and RNA pulldown experiment were used to evaluate the interaction of NR_030777 with its target proteins. NR_030777 and mitophagy were increased, and tyrosine hydroxylase (TH) levels recovered after NR_030777 overexpression upon PQ treatment. The overexpression and knockdown of NR_030777 unveiled that NR_030777 positively regulated mitophagy such as the upregulation of LC3B-II:I, ATG12-ATG5, p62 and NBR1. Moreover, the application of mdivi-1, a DRP-1 inhibitor, in combination with NR_030777 genetic modified cells unveiled that NR_030777 promoted DRP1-mediated mitochondrial fission and mitophagy. Furthermore, NR_030777 were directly bound to CDK1 to increase p-DRP1 levels at the Ser616 site, leading to mitochondrial fission and mitophagy. On the other hand, NR_030777 acted directly on ATG12 within the ATG12-ATG5 complex in the 800-1400 nt region to modulate the membrane formation. Accordingly, NR_030777 deficiency in neuron cells compromised cell mitophagy. Finally, the above findings were confirmed using NR_030777-overexpressing mice. NR_030777 exerted a protective effect on PQ-exposed mice by enhancing mitophagy. Our data provide the first scientific evidence for the precise invention of PQ-induced PD. Our findings further propose a breakthrough for understanding the regulatory relationship between NR_030777, CDK1, ATG12 and mitophagy in PQ-induced PD.

Single-cell RNA-sequencing of cellular heterogeneity and pathogenic mechanisms in paraquat-induced Parkinson's disease with depression.

Parkinson's disease (PD) is among the most prevalent neurodegenerative diseases, and approximately one third of patients with PD are estimated to have depression. Paraquat (PQ) exposure is an important environmental risk factor for PD. In this study, we established a mouse model of PQ-induced PD with depression to comprehensively investigate cellular heterogeneity and the mechanisms underlying the progression of depression in the context of PD. We utilized single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of individual cells from model mice and characterize the gene expression profiles in each differentially expressed cell type. We identified a specific glutamatergic neuron cluster responsible for the development of heterogeneous depression-associated changes and established a comprehensive gene expression atlas. Furthermore, functional enrichment and cell trajectory analyses revealed that the mechanisms underlying the progression of PD with depression were associated with specific glutamatergic neurons. Together, our findings provide a valuable resource for deciphering the cellular heterogeneity of PD with depression. The suggested connection between intrinsic transcriptional states of neurons and the progression of depression can provide insight into potential biomarkers and specific targets for anti-depression treatment in patients with PD. SYNOPSIS: Our results obtained using model mice confirm the core effects of PQ exposure on glutamatergic neurons and their potential role in the development of PD with depression.

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.

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.

Cobalt induces neurodegeneration through FTO-triggered autophagy impairment by targeting TSC1 in an m6A-YTHDF2-dependent manner.

Cobalt is the most widely used heavy metal pollutant in medicine and industry. Excessive cobalt exposure can adversely affect human health. Neurodegenerative symptoms have been observed in cobalt-exposed populations; however, the underlying mechanisms remain largely unknown. In this study, we demonstrate that the N6-methyladenosine (m6A) demethylase fat mass and obesity-associated gene (FTO) mediates cobalt-induced neurodegeneration by impairing autophagic flux. Cobalt-induced neurodegeneration was exacerbated through FTO genetic knockdown or repression of demethylase activity, but was alleviated by FTO overexpression. Mechanistically, we showed that FTO regulates TSC1/2-mTOR signaling pathway by targeting TSC1 mRNA stability in an m6A-YTHDF2 manner, which resulted in autophagosome accumulation. Furthermore, FTO decreases lysosome-associated membrane protein-2 (LAMP2) to inhibit the integration of autophagosomes and lysosomes, leading to autophagic flux damage. In vivo experiments further identified that central nervous system (CNS)-Fto-specific knockout resulted in serious neurobehavioral and pathological damage as well as TSC1-related autophagy impairment in cobalt-exposed mice. Interestingly, FTO-regulated autophagy impairment has been confirmed in patients with hip replacement. Collectively, our results provide novel insights into m6A-modulated autophagy through FTO-YTHDF2 targeted TSC1 mRNA stability, revealing cobalt is a novel epigenetic hazard that induces neurodegeneration. These findings suggest the potential therapeutic targets for hip replacement in patients with neurodegenerative damage.

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.

Cobalt induces neurodegenerative damages through impairing autophagic flux by activating hypoxia-inducible factor-1α triggered ROS overproduction.

Cobalt is an environmental toxicant, and excessive bodily exposure can damage the nervous system. Particularly, our previous study reported that low-dose cobalt (significantly less than the safety threshold) is still able to induce neurodegenerative changes. However, the underlying molecular mechanism is still insufficient revealed. Herein, we further investigate the molecular mechanism between cobalt-induced neurodegeneration and autophagy, as well as explore the interplay between hypoxia-inducible factor-1α (HIF-1α), reactive oxygen species (ROS), and autophagy in cobalt-exposed mice and human neuroglioma cells. We first reveal cobalt as an environmental toxicant to severely induce ß amyloid (Aß) deposition, tau hyperphosphorylation, and dysregulated autophagy in the hippocampus and cortex of mice. In particular, we further identify that cobalt-induced neurotoxicity is triggered by the impairment of autophagic flux in vitro experiments. Moreover, the mechanistic study reveals that cobalt exposure extremely activates HIF-1α expression to facilitate the overproduction of ROS. Then, elevated ROS can target the amino-threonine kinase (AKT)-mammalian target of rapamycin (mTOR)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling pathway to participate in cobalt-induced impairment of autophagic flux. Subsequently, defected autophagy further exacerbates cobalt-induced neurotoxicity for its unable to eliminate the deposition of pathological protein. Therefore, our data provide scientific evidence for cobalt safety evaluation and risk assessment and propose a breakthrough for understanding the regulatory relationship between HIF-1α, ROS, and autophagy in cobalt-induced neurodegeneration.

4-Mercaptobenzoic acid-assisted laser desorption/ionization mass spectrometry for sensitive quantification of cesium and strontium in drinking water.

RATIONALE: Cesium ions (Cs+ ) and strontium ions (Sr2+ ), which enter the human body mainly through drinking water, are an important determinant of health. They are widely distributed on Earth and extremely soluble in water. In order to assist assessment of the drinking safety, it was essential to develop a rapid analytical method for quantification. We have established a 4-mercaptobenzoic acid (MBA)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method for the rapid detection and sensitive quantification of Cs+ and Sr2+ in the aqueous environment. METHODS: Using MBA as the matrix, the rapid detection and quantification for Cs+ and Sr2+ were conducted by MALDI-TOF-MS. At first, the concentration of MBA was optimized. Then, salt tolerance, detection limit and reproducibility of this method were evaluated by standard solutions. Finally, the calibration curves were constructed and applied to the rapid determination of Cs+ and Sr2+ in six commercially available bottled waters for drinking. RESULTS: For the MBA-assisted MALDI-TOF-MS method, the optimal concentration of MBA was 2 mg/mL. The signal-to-noise (S/N) ratio of Cs+ was up to 971 in 1000 mmol/mL NaCl solution. The detection limits of the method for Cs+ and Sr2+ were 3 pg/mL and 10 pg/mL, respectively. Furthermore, this developed method was applied to the rapid analysis of Cs+ and Sr2+ in six commercially available drinking waters, and the results correlated well with the results obtained from a validated inductively coupled plasma mass spectrometry (ICP-MS) method. CONCLUSIONS: The MBA-assisted MALDI-TOF-MS method has high sensitivity, fast detection speed, less background interference, and high reproducibility in the analysis of Cs+ and Sr2+ . Because of the physiological functions and general toxic effects, detection of Cs+ and Sr2+ in water is of major importance for drinking safety.

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.

Covalent organic framework-based solid phase microextraction coupled with electrospray ionization mass spectrometry for sensitive screening and quantitative evaluation of carbamazepine and its metabolite in mice.

Carbamazepine (CBZ) and its metabolite carbamazepine-10,11-epoxide (CBZEP) play vital role in the treatment of epilepsy. It is of great importance to develop a method for rapid and sensitive monitoring of CBZ and CBZEP due to their narrow therapeutic index. Herein, an imine-linked-based covalent organic framework was synthesized by using 1,3,5-tris (4-aminophenyl) benzene (TPB) and 1,3,5-triformylbenzene (TFB) (denoted as TPB-TFB-COF)，and applied as a solid-phase microextraction (SPME) probe for extracting CBZ and CBZEP. The TPB-TFB-COF showed large surface areas (371 m2 g-1), high regular porosity (1.23 nm) and extraordinary stability, which rendered it an ideal adsorbent for highly efficient enrichment of CBZ and CBZEP. Accordingly, an attractive strategy of the combination of the TPB-TFB-COF-based SPME probe and electrospray ionization mass spectrometry system (ESI/MS) was proposed for rapid screening and sensitive monitoring of CBZ and CBZEP. Under the optimized parameters, the developed method exhibited good linearity for CBZ and CBZEP in the range of 4-1000 µg L-1 with correlation coefficient (r) no less than 0.9953, and the corresponding limits of detection (LODs) were 0.4 and 2.5 µg L-1, respectively. Moreover, high enrichment factors (EFs, 202-351 folds) and satisfactory relative standard deviations (RSDs) of one probe (3.3-5.1%) and probe-to-probe (4.8-5.6%) were obtained. By using the proposed method, sensitive screening and quantitative evaluation of CBZ and CBZEP in mice whole blood and tissue homogenates were successfully achieved, indicating the promising applicability of the TPB-TFB-COF-SPME-AMIS as a powerful tool for drug monitoring.

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 Application of Omics Technologies in the Research of Neurotoxicology.

Omics technologies offer unprecedented perspectives for the systematic research of complex organisms. Especially, the comprehensive omics provide powerful tools for exploring extensive perception of the biochemical and/or physiological effects of the cells perturbed by poisons through systemic investigation on the different spatiotemporal expression profiles of transcriptomic expression profiling, expressed proteins and metabolites simultaneously by high-throughput and high-sensitivity technology. Therefore, it has been attracted extensive attention in neurotoxicology. During the recent years, the domain of neurotoxicology has progressively assimilated various emerging omics applications, which mainly include transcriptomics, proteomics, and metabolomics, to investigate the molecular mechanisms and potential molecular biomarkers of phenotypic modifications of organisms subjected to poisons or stress factors.

Cobalt induces neurodegenerative damages through Pin1 inactivation in mice and human neuroglioma cells.

Cobalt is a hazardous material that has harmful effects on neurotoxicity. Excessive exposure to cobalt or inactivation of the unique proline isomerase Pin1 contributes to age-dependent neurodegeneration. However, nothing is known about the role of Pin1 in cobalt-induced neurodegeneration. Here we find that out of several hazardous materials, only cobalt dose-dependently decreased Pin1 expression and alterations in its substrates, including cis and trans phosphorylated Tau in human neuronal cells, concomitant with neurotoxicity. Cobalt-induced neurotoxicity was aggravated by Pin1 genetic or chemical inhibition, but rescued by Pin1 upregulation. Furthermore, less than 4 µg/l of blood cobalt induced dose- and age-dependent Pin1 downregulation in murine brains, ensuing neurodegenerative changes. These defects were corroborated by changes in Pin1 substrates, including cis and trans phosphorylated Tau, amyloid precursor protein, ß amyloid and GSK3ß. Moreover, blood Pin1 was downregulated in human hip replacement patients with median blood cobalt level of 2.514 µg/l, which is significantly less than the safety threshold of 10 µg/l, suggesting an early role Pin1 played in neurodegenerative damages. Thus, Pin1 inactivation by cobalt contributes to age-dependent neurodegeneration, revealing that cobalt is a hazardous material triggering AD-like neurodegenerative damages.

Drp1-mediated mitochondrial fission contributes to mitophagy in paraquat-induced neuronal cell damage.

Paraquat (PQ) is one of the most widely used herbicides in the world due to its excellent weed control effects. Accumulating evidence has revealed that long-term exposure to PQ can significantly increase the risk of Parkinson's disease (PD). However, the underlying molecular mechanisms are yet to be fully understood. Hence, we investigated the potential role of reactive oxygen species (ROS) and dynamin-related protein 1 (DRP1) in PQ-induced mitophagy, aiming to elaborate on possible molecular mechanisms involved in PQ-triggered neurotoxicity. Our results showed that ROS were increased, mitochondrial membrane potential was decreased at 100, 200, and 300 µM PQ concentrations, and autophagy pathways were activated at a concentration of 100 µM in neuronal cells. In addition, excessive mitophagy was observed in neurons exposed to 300 µM PQ for 24 h. Then, ROS-mediated mitochondrial fission was found to contribute to PQ-induced excessive mitophagy. Moreover, all aforementioned changes were significantly ameliorated by mdivi-1. Thus, our findings provide a novel neurotoxic mechanism and reveal the DRP1-mitochondrial fission pathway as a potential target for treatments of PQ-induced excessive mitophagy, serving as an alternative target for the prevention and treatment of Parkinson's disease. Because harmful substances are transmitted and enriched in the food chain, the toxic effect of environmental paraquat is nonnegligible, and more investigations are needed.