Yan Sai

Dopaminergic neurodegeneration constitutes the core pathological feature of Parkinson’s disease (PD). Rotenone exposure triggers dopaminergic neuronal damage and contributes to PD pathogenesis. In this study, in-vivo and in-vitro models of rotenone exposure were established. Multiple inhibitors were used for intervention, and experimental techniques such as Western blot, qPCR, ELISA, and CO-IP were used. The release of mtDNA induced by rotenone exposure with qPCR. The activation of cytoplasmic cGAS-STING, NLRP3-Caspase 1- GSDMD-IL-1β signaling pathways were detected with Western blot, ELISA and so on. The related pathways of mtDNA release were detected with CO-IP and molecular docking. Meanwhile, the effects of rotenone exposure on the content of NAD+, key synthesis enzymes, and transport carriers were examined. The study explored the impact of rotenone on NAD+ metabolism in dopaminergic neurons, as well as the effect of exogenous NAD+ supplementation in improving pyroptotic damage caused by rotenone-induced mtDNA release by regulating the opening state of mPTP. The present study demonstrated that rotenone disrupts NAD⁺ metabolism, which induces excessive mPTP opening and promotes GSDMDNT oligomerization on mitochondrial membranes, ultimately resulting in mtDNA leakage into the cytosol. Cytosolic mtDNA further activates the cGASSTING–NLRP3–caspase-1 signaling cascade and drives neuronal pyroptosis in PC12 cells. Exogenous NAD⁺ pretreatment stabilizes mPTP and restricts mtDNA release through two coordinated mechanisms: (i) SIRT3mediated deacetylation of mPTP components, including ANT1 and CypD; and (ii) direct competitive binding of NAD⁺ to ANT1 to block CypD–ANT1 interaction and limit mPTP opening. Accordingly, NAD⁺ supplementation alleviates rotenoneinduced dopaminergic neurodegeneration by inhibiting mtDNA release and suppressing cGASSTING/NLRP3 inflammatory signaling. NAD⁺ targets mPTP and GSDMDNT to interrupt the mtDNA–inflammation–pyroptosis axis, providing a promising therapeutic strategy for neurodegeneration driven by rotenonemediated mtDNA release.

Objective: Mitochondrial dysfunction is the key factor in rotenone-induced neurotoxicity in dopaminergic neurons. This study aimed to investigate the role and potential mechanism of the mitochondrial DNA encoded peptide Humanin (HN) in alleviating rotenone-induced neurotoxicity. Methods: Rotenone was added to the cultured PC12 cells to induce neurotoxicity. PC12 cells were preincubated with HN, which has a protective effect. Cell counting kit-8 (CCK-8) was used to evaluate PC12 cell viability. Flow cytometry to detect the content of reactive oxygen species (ROS) in PC12 cells. Western blot analysis was used to detect the expression of superoxide dismutase 2 (SOD2), acetylated SOD (Ac-SOD), sirtuin 3 (SIRT3), nuclear factor erythroid 2-related factor 2 (Nrf2), heme-oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1). The corresponding kits were used to measure the NAD+/NADH ratio and SOD content separately. Results: HN pretreatment significantly increased PC12 cell survival, reduced ROS formation, and increased the NAD ⁺ /NADH ratio. It also increased the expression of SIRT3, Nrf2, HO-1, and NQO1 proteins and decreased the expression of Ac-SOD protein under rotenone exposure. At the same time, it also activated the Nrf2/HO-1 signaling pathway, which depends on HN-mediated SIRT3 activation. Conclusion: These results suggest that HN plays a protective role in rotenone-induced neurotoxicity by suppressing oxidative stress and activating the antioxidant response via the Nrf2/HO-1 pathway, which is regulated by SIRT3 in PC12 cells.

Parkinson's disease is a neurodegenerative disorder whose pathogenesis remains incompletely understood. Rotenone exposure is reportedly associated with Parkinson's disease. In addition, disulfidptosis is a newly identified form of cell death. Interestingly, an analysis of the Gene Expression Omnibus Parkinson's disease database indicated that approximately 30 genes that are significantly altered in patients with Parkinson's disease are associated with disulfidptosis. In the present study, using proteomics, a number of important proteins related to disulfidptosis were identified as significantly altered in rotenone-exposed dopaminergic neurons. Further analysis revealed that the formation of abnormal disulfide bonds was also increased in rotenone-exposed dopaminergic neurons. The protein expression of solute carrier family 7 member 11 and amino acid transporter heavy chain SLC3A2 was upregulated in rotenone-exposed dopaminergic neurons, and was correlated with extracellular matrix protein 1 protein expression. These findings indicate that in rotenone-exposed PC12 cells, a cystine influx is triggered, and the conversion of cystine to cysteine is inhibited by a reduction in the oxidized nicotinamide adenine dinucleotide phosphate/reduced nicotinamide adenine dinucleotide phosphate ratio, which leads to cystine accumulation. This excessive accumulation of cystine then promotes the formation of abnormal disulfide bonds in cells, ultimately resulting in disulfidptosis of rotenone-exposed dopaminergic neurons. In this process, the Rasrelated C3 botulinum toxin substrate 1/WAVE regulatory complex/actin-related protein 2/3 pathway was markedly activated, which led to the collapse of the cytoskeleton in rotenone-exposed PC12 cells. Together, our findings suggest that rotenone may induce solute carrier family 7 member 11 expression through extracellular matrix protein 1 activation to cause cystine accumulation, which results in disulfidptosis characterized by cytoskeleton collapse. The present results provide new perspectives for research into neurodegenerative diseases.

MOTS-c is a 16-amino acid mitochondrial-derived peptide reported to be involved in regulating energy metabolism. However, few studies have reported the role of MOTS-c on neuron degeneration. In this study, it was aimed to explore the action of MOTS-c in rotenone-induced dopaminergic neurotoxicity. In an in vitro study, it was observed that rotenone could influence the expression and localization of MOTS-c significantly in PC12 cells, with more MOTS-c translocating into the nucleus from mitochondria. Further study showed that the translocation of MOTS-c from the mitochondria into the nucleus could directly interact with Nrf2 to regulate HO-1 and NQO1 expression in PC12 cells exposed to rotenone, which had been suggested to be involved in the antioxidant defense system. In vivo and in vitro experiments demonstrated that exogenous MOTS-c pretreatment could protect PC12 cells and rats from mitochondrial dysfunction and oxidative stress induced by rotenone. Moreover, MOTS-c pretreatment significantly decreased the loss of TH, PSD95, and SYP protein expression in the striatum of rats exposed to rotenone. In addition, MOTS-c pretreatment could clearly alleviate the downregulated expression of Nrf2, HO-1, and NQO1, as well as the upregulated Keap1 protein expression in the striatum of rotenone-treated rats. Taken together, these findings suggested that MOTS-c could directly interact with Nrf2 to activate the Nrf2/HO-1/NQO1 signal pathway to defend the antioxidant system to prevent dopaminergic neurons from rotenone-induced oxidative stress and neurotoxicity in vitro and in vivo.

: Sulfur mustard (SM), a classic chemical weapon in the vesicant category, can induce severe damage, for which the therapy is still limited even today. Laboratory work is essential in unveiling toxicological effects and developing medical countermeasures. Sulfur mustard analog 2-chloroethyl ethyl sulfide (CEES), is employed in the lab for less toxicity. However, due to its similar characteristics to SM (being oily, hydrophobic, and volatile), the manipulation of CEES still needs special attention to avoid personnel injury and laboratory pollution. Here, to clear the chemical safety concerns in the laboratory study of CEES, the working procedure and experimental data are summarized, which might help educate new researchers to be skilled and professional.

Mitochondria harbor small circular genomes (mtDNA) that encode 13 oxidative phosphorylation (OXPHOS) proteins, and types of damage to mtDNA may contribute to neuronal damage. Recent studies suggested that regulation of mtDNA repair proteins may be a potential strategy for treating neuronal damage. The mtDNA repair system contains its own repair enzymes and is independent from the nuclear DNA repair system. Endo/exonuclease G-like(EXOG) is a mitochondria-specific 5-exo/endonuclease required for repairing endogenous single-strand breaks (SSBs) in mtDNA. However, whether EXOG plays a key role in neuronal damage induced by rotenone remains unknown. Thus, in this study, we aimed to investigate the effect of EXOG on mtDNA repair and mitochondrial functional maintenance in rotenone-induced neurotoxicity. Our results indicated that rotenone influenced the expression and location of EXOG in PC12 cells. Meanwhile, after rotenone exposure, the expression was reduced for proteins responsible for mtDNA repair, including DNA polymerase γ (POLG), high-temperature requirement protease A2 (HtrA2), and the heat-shock factor 1-single-stranded DNA-binding protein 1 (HSF1-SSBP1) complex. Further analysis demonstrated that EXOG knockdown led to reduced mtDNA copy number and mtDNA transcript level and increased mtDNA deletion, which further aggravated the mtDNA damage and mitochondrial dysfunction under rotenone stress. In turn, EXOG overexpression protected PC12 cells from mtDNA damage and mitochondrial dysfunction induced by rotenone. As a result, EXOG knockdown reduced cell viability and tyrosine hydroxylase expression, while EXOG overexpression alleviated rotenone’s effect on cell viability and tyrosine hydroxylase expression in PC12 cells. Further, we observed that EXOG influenced mtDNA repair by regulating protein expression of the HSF1-SSBP1 complex and POLG. Furthermore, our study showed that PGC-1α upregulation with ZLN005 led to increased protein levels of EXOG, POLG, HSF1, and SSBP1, all of which contribute to mtDNA homeostasis. Therefore, PGC-1α may be involved in mtDNA repair through interacting with multiple mtDNA repair proteins, especially with the help of EXOG. In summary, EXOG regulation by PGC-1α plays an essential role in rotenone-induced neurotoxicity in PC12 cells. EXOG represents a protective effect strategy in PC12 cells exposed to rotenone.

Background: Medical defense against chemical, biological, radiological, and nuclear (CBRN) threats should be established through systematic education in college and career development training to counter the response successfully. Methods: Taking the course of Medical Countermeasures against CBRN Weapons is compulsorily for all military medical students in the Chinese People’s Liberation Army (PLA). In the present study, a modularized course for field training of Chemical Defense Medicine is provided, which is an important part of the Integrated Military Medicine Exercise (IMME) conducted annually in PLA military medical university. Discussion: The established modules are functionally designed as passing through a contaminated area with protective posture, operating chemical defense equipment, battlefield rescue after a chemical attack, and early treatment of chemically contaminated casualties. This modularized training is task-oriented, function-based, and integrate with a functional drill, which provides an example in the training of CBRN defense medicine.