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.

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.

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.

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.

Ferroptosis contributes to hemolytic hyperbilirubinemia­induced brain damage in vivo and in vitro.

Ferroptosis is driven by iron­dependent accumulation of lipid hydroperoxides, and hemolytic hyperbilirubinemia causes accumulation of unconjugated bilirubin and iron. The present study aimed to assess the role of ferroptosis in hemolytic hyperbilirubinemia­induced brain damage (HHIBD). Rats were randomly divided into the control, phenylhydrazine (PHZ) and deferoxamine (DFO) + PHZ groups, with 12 rats in each group. Ferroptosis­associated biochemical and protein indicators were measured in the brain tissue of rats. We also performed tandem mass tag­labeled proteomic analysis. The levels of iron and malondialdehyde were significantly higher and levels of glutathione (GSH) and superoxide dismutase activity significantly lower in the brain tissues of the PHZ group compared with those in the control group. HHIBD also resulted in significant increases in the expression of the ferroptosis­related proteins acyl­CoA synthetase long­chain family member 4, ferritin heavy chain 1 and transferrin receptor and divalent metal transporter 1, as well as a significant reduction in the expression of ferroptosis suppressor protein 1. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that the differentially expressed proteins of rat brain tissues between the control and PHZ groups were significantly involved in ferroptosis, GSH metabolism and fatty acid biosynthesis pathways. Pretreatment with DFO induced antioxidant activity and alleviated lipid peroxidation­mediated HHIBD. In addition, PC12 cells treated with ferric ammonium citrate showed shrinking mitochondria, high mitochondrial membrane density, and increased lipid reactive oxygen species and intracellular ferrous iron, which were antagonized by pretreatment with ferrostatin­1 or DFO, which was reversed by pretreatment with ferrostatin­1 or DFO. The present study demonstrated that ferroptosis is involved in HHIBD and provided novel insights into candidate proteins that are potentially involved in ferroptosis in the brain during hemolytic hyperbilirubinemia.

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 deficiency of N6-methyladenosine demethylase ALKBH5 enhances the neurodegenerative damage induced by cobalt.

Cobalt exposure, even at low concentrations, induces neurodegenerative damage, such as Alzheimer's disease (AD). The specific underlying mechanisms remain unclear. Our previous study demonstrated that m6A methylation alteration is involved in cobalt-induced neurodegenerative damage, such as in AD. However, the role of m6A RNA methylation and its underlying mechanisms are poorly understood. In this study, both epidemiological and laboratory studies showed that cobalt exposure could downregulate the expression of the m6A demethylase ALKBH5, suggesting a key role for ALKBH5. Moreover, Methylated RNA immunoprecipitation and sequencing (MeRIP-seq) analysis revealed that ALKBH5 deficiency is associated with neurodegenerative diseases. KEGG pathway and Gene ontology analyses further revealed that the differentially m6A-modified genes resulting from ALKBH5 downregulation and cobalt exposure were aggregated in the pathways of proliferation, apoptosis, and autophagy. Subsequently, ALKBH5 deficiency was shown to exacerbate cell viability decline, motivate cell apoptosis and attenuate cell autophagy induced by cobalt with experimental techniques of gene overexpression/inhibition. In addition, morphological changes in neurons and the expression of AD-related proteins, such as APP, P-Tau, and Tau, in the cerebral hippocampus of wild-type and ALKBH5 knockout mice after chronic cobalt exposure were also investigated. Both in vitro and in vivo results showed that lower expression of ALKBH5 aggravated cobalt-induced neurodegenerative damage. These results suggest that ALKBH5, as an epigenetic regulator, could be a potential target for alleviating cobalt-induced neurodegenerative damage. In addition, we propose a novel strategy for the prevention and treatment of environmental toxicant-related neurodegeneration from an epigenetic perspective.

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 nanoparticles induce mitochondrial damage and ß-amyloid toxicity via the generation of reactive oxygen species.

Exposure to cobalt nanoparticles (CoNPs) has been associated with neurodegenerative disorders, while the mitochondrial-associated mechanisms that mediate their neurotoxicity have yet to be fully characterized. In this study, we reported that CoNPs exposure reduced the survival and lifespan in the nematodes, Caenorhabditis elegans (C. elegans). Moreover, exposure to CoNPs aggravated the induction of paralysis and the aggregation of ß-amyloid (Aß). These effects were accompanied by reactive oxygen species (ROS) overproduction, ATP reduction as well as mitochondrial fragmentation. Dynamin-related protein 1 (drp-1) activation and ensuing mitochondrial fragmentation have been shown to be associated with CoNPs-reduced survival. In order to address the role of mitochondrial damage and ROS production in CoNPs-induced Aß toxicity, the mitochondrial reactive oxygen species scavenger mitoquinone (Mito Q) was used. Our results showed that Mito Q pretreatment alleviated CoNPs-induced ROS generation, rescuing mitochondrial dysfunction, thereby lessening the CoNPs-induced Aß toxicity. Taken together, we show for the first time, that increasing of ROS and the upregulation of drp-1 lead to CoNPs-induced Aß toxicity. Our novel findings provide in vivo evidence for the mechanisms of environmental toxicant-induced Aß toxicity, and can afford new modalities for the prevention and treatment of CoNPs-induced neurodegeneration.

Intercellular transfer of mitochondria via tunneling nanotubes protects against cobalt nanoparticle-induced neurotoxicity and mitochondrial damage.

Broad applications of cobalt nanoparticles (CoNPs) have raised increased concerns regarding their potential toxicity. However, the underlining mechanisms of their toxicity have yet to be characterized. Here, we demonstrated that CoNPs reduced cell viability and induced membrane leakage. CoNPs induced oxidative stress, as indicated by the generation of reactive oxygen species (ROS) secondary to the increased expression of hypoxia-induced factor 1 alpha. Moreover, CoNPs led to mitochondrial damage, including generation of mitochondrial ROS, reduction in ATP content, morphological damage and autophagy. Interestingly, exogenous mitochondria were observed between neurons and astrocytes upon CoNPs exposure. Concomitantly, tunneling nanotubes (TNTs)-like structures were observed between neurons and astrocytes upon CoNPs exposure. These structures were further verified to be TNTs as they were found to be F-actin rich and lacking tubulin. We then demonstrated that TNTs were utilized for mitochondrial transfer between neurons and astrocytes, suggesting a novel crosstalk phenomenon between these cells. Moreover, we found that the inhibition of TNTs (using actin-depolymerizing drug latrunculin B) intensified apoptosis triggered by CoNPs. Therefore, we demonstrate, for the first time, that the inhibition of intercellular mitochondrial transfer via TNTs aggravates CoNPs-induced cellular and mitochondrial toxicity in neuronal cells, implying a novel intercellular protection mechanism in response to nanoparticle exposure.