Novel Thienopyrimidine-Hydrazinyl Compounds Induce DRP1-Mediated Non-Apoptotic Cell Death in Triple-Negative Breast Cancer Cells.

Apoptosis induction with taxanes or anthracyclines is the primary therapy for TNBC. Cancer cells can develop resistance to anticancer drugs, causing them to recur and metastasize. Therefore, non-apoptotic cell death inducers could be a potential treatment to circumvent apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induced non-apoptotic cell death in TNBC cells. These lead compounds were 15- to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to that of normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death, characterized by the absence of cellular shrinkage and the absence of nuclear fragmentation and apoptotic blebs. Although TPH104c and TPH104m induced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase the levels of cytochrome c and intracellular reactive oxygen species (ROS) and caspase activation, and cell death was not rescued by incubating cells with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). Furthermore, TPH104c and TPH104m significantly downregulated the expression of the mitochondrial fission protein, DRP1, and their levels determined their cytotoxic efficacy. Overall, TPH104c and TPH104m induced non-apoptotic cell death, and further determination of their cell death mechanisms will aid in the development of new potent and efficacious anticancer drugs to treat TNBC.

The Effect of Cold-Water Swimming on Energy Metabolism, Dynamics, and Mitochondrial Biogenesis in the Muscles of Aging Rats.

Our study aimed to explore the potential positive effects of cold water exercise on mitochondrial biogenesis and muscle energy metabolism in aging rats. The study involved 32 male and 32 female rats aged 15 months, randomly assigned to control sedentary animals, animals training in cold water at 5 ± 2 °C, or animals training in water at thermal comfort temperature (36 ± 2 °C). The rats underwent swimming training for nine weeks, gradually increasing the duration of the sessions from 2 min to 4 min per day, five days a week. The results demonstrated that swimming in thermally comfortable water improved the energy metabolism of aging rat muscles (increased metabolic rates expressed as increased ATP, ADP concentration, TAN (total adenine nucleotide) and AEC (adenylate energy charge value)) and increased mRNA and protein expression of fusion regulatory proteins. Similarly, cold-water swimming improved muscle energy metabolism in aging rats, as shown by an increase in muscle energy metabolites and enhanced mitochondrial biogenesis and dynamics. It can be concluded that the additive effect of daily activity in cold water influenced both an increase in the rate of energy metabolism in the muscles of the studied animals and an intensification of mitochondrial biogenesis and dynamics (related to fusion and fragmentation processes). Daily activity in warm water also resulted in an increase in the rate of energy metabolism in muscles, but at the same time did not cause significant changes in mitochondrial dynamics.

The role of DRP1 mediated mitophagy in HT22 cells apoptosis induced by silica nanoparticles.

Silica nanoparticles (SiNPs) are widely used in the biomedical field and can enter the central nervous system through the blood-brain barrier, causing damage to hippocampal neurons. However, the specific mechanism remains unclear. In this experiment, HT22 cells were selected as the experimental model in vitro, and the survival rate of cells under the action of SiNPs was detected by MTT method, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and adenosine triphosphate (ATP) were tested by the kit, the ultrastructure of the cells was observed by transmission electron microscope, membrane potential (MMP), calcium ion (Ca2+) and apoptosis rate were measured by flow cytometry, and the expressions of mitochondrial functional protein, mitochondrial dynein, mitochondrial autophagy protein as well as apoptosis related protein were detected by Western blot. The results showed that cell survival rate, SOD, CAT, GSH-Px, ATP and MMP gradually decreased with the increase of SiNPs concentration, while intracellular ROS, Ca2+, LDH and apoptosis rate increased with the increase of SiNPs concentration. In total cellular proteins,the expressions of mitochondrial functional proteins VDAC and UCP2 gradually increased, the expression of mitochondrial dynamic related protein DRP1 increased while the expressions of OPA1 and Mfn2 decreased. The expressions of mitophagy related proteins PINK1, Parkin and LC3Ⅱ/LC3Ⅰ increased and P62 gradually decreased, as well as the expressions of apoptosis related proteins Apaf-1, Cleaved-Caspase-3, Caspase-3, Caspase-9, Bax and Cyt-C. In mitochondrial proteins, the expressions of mitochondrial dynamic related proteins DRP1 and p-DRP1 were increased, while the expressions of OPA1 and Mfn2 were decreased. Expressions of mitochondrial autophagy associated proteins PINK1, Parkin, LC3II/LC3I increased, P62 decreased gradually, as well as the expressions of apoptosis related proteins Cleaved-Caspase-3, Caspase-3, and Caspase-9 increased, and Cyt-C expressions decreased. To further demonstrate the role of ROS and DRP1 in HT22 cell apoptosis induced by SiNPs, we selected the ROS inhibitor N-Acetylcysteine (NAC) and Dynamin-related protein 1 (DRP1) inhibitor Mdivi-1. The experimental results indicated that the above effects were remarkably improved after the use of inhibitors, further confirming that SiNPs induce the production of ROS in cells, activate DRP1, cause excessive mitochondrial division, induce mitophagy, destroy mitochondrial function and eventually lead to apoptosis.

Multifaceted functions of Drp1 in hypoxia/ischemia-induced mitochondrial quality imbalance: from regulatory mechanism to targeted therapeutic strategy.

Hypoxic-ischemic injury is a common pathological dysfunction in clinical settings. Mitochondria are sensitive organelles that are readily damaged following ischemia and hypoxia. Dynamin-related protein 1 (Drp1) regulates mitochondrial quality and cellular functions via its oligomeric changes and multiple modifications, which plays a role in mediating the induction of multiple organ damage during hypoxic-ischemic injury. However, there is active controversy and gaps in knowledge regarding the modification, protein interaction, and functions of Drp1, which both hinder and promote development of Drp1 as a novel therapeutic target. Here, we summarize recent findings on the oligomeric changes, modification types, and protein interactions of Drp1 in various hypoxic-ischemic diseases, as well as the Drp1-mediated regulation of mitochondrial quality and cell functions following ischemia and hypoxia. Additionally, potential clinical translation prospects for targeting Drp1 are discussed. This review provides new ideas and targets for proactive interventions on multiple organ damage induced by various hypoxic-ischemic diseases.

The Role of Mitochondrial Dynamics and Mitotic Fission in Regulating the Cell Cycle in Cancer and Pulmonary Arterial Hypertension: Implications for Dynamin-Related Protein 1 and Mitofusin2 in Hyperproliferative Diseases.

Mitochondria, which generate ATP through aerobic respiration, also have important noncanonical functions. Mitochondria are dynamic organelles, that engage in fission (division), fusion (joining) and translocation. They also regulate intracellular calcium homeostasis, serve as oxygen-sensors, regulate inflammation, participate in cellular and organellar quality control and regulate the cell cycle. Mitochondrial fission is mediated by the large GTPase, dynamin-related protein 1 (Drp1) which, when activated, translocates to the outer mitochondrial membrane (OMM) where it interacts with binding proteins (Fis1, MFF, MiD49 and MiD51). At a site demarcated by the endoplasmic reticulum, fission proteins create a macromolecular ring that divides the organelle. The functional consequence of fission is contextual. Physiological fission in healthy, nonproliferating cells mediates organellar quality control, eliminating dysfunctional portions of the mitochondria via mitophagy. Pathological fission in somatic cells generates reactive oxygen species and triggers cell death. In dividing cells, Drp1-mediated mitotic fission is critical to cell cycle progression, ensuring that daughter cells receive equitable distribution of mitochondria. Mitochondrial fusion is regulated by the large GTPases mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2), which fuse the OMM, and optic atrophy 1 (OPA-1), which fuses the inner mitochondrial membrane. Mitochondrial fusion mediates complementation, an important mitochondrial quality control mechanism. Fusion also favors oxidative metabolism, intracellular calcium homeostasis and inhibits cell proliferation. Mitochondrial lipids, cardiolipin and phosphatidic acid, also regulate fission and fusion, respectively. Here we review the role of mitochondrial dynamics in health and disease and discuss emerging concepts in the field, such as the role of central versus peripheral fission and the potential role of dynamin 2 (DNM2) as a fission mediator. In hyperproliferative diseases, such as pulmonary arterial hypertension and cancer, Drp1 and its binding partners are upregulated and activated, positing mitochondrial fission as an emerging therapeutic target.

YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

Cancer stem cells (CSCs) are associated with tumor progression, recurrence, and therapeutic resistance. To maintain their pool while promoting tumorigenesis, CSCs divide asymmetrically, producing a CSC and a highly proliferative, more differentiated transit-amplifying cell. Exhausting the CSC pool has been proposed as an effective antitumor strategy; however, the mechanism underlying CSC division remains poorly understood, thereby largely limiting its clinical application. Here, through cross-omics analysis, yin yang 2 (YY2) is identified as a novel negative regulator of CSC maintenance. It is shown that YY2 is downregulated in stem-like tumor spheres formed by hepatocarcinoma cells and in liver cancer, in which its expression is negatively correlated with disease progression and poor prognosis. Furthermore, it is revealed that YY2 overexpression suppressed liver CSC asymmetric division, leading to depletion of the CSC pool and decreased tumor-initiating capacity. Meanwhile, YY2 knock-out in stem-like tumor spheres caused enrichment in mitochondrial functions. Mechanistically, it is revealed that YY2 impaired mitochondrial fission, and consequently, liver CSC asymmetric division, by suppressing the transcription of dynamin-related protein 1. These results unravel a novel regulatory mechanism of mitochondrial dynamic-mediated CSCs asymmetric division and highlight the role of YY2 as a tumor suppressor and a therapeutic target in antitumor treatment.

Mitochondrial impairment and downregulation of Drp1 phosphorylation underlie the antiproliferative and proapoptotic effects of alantolactone on oral squamous cell carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most prevalent and fatal oral cancers. Mitochondria-targeting therapies represent promising strategies against various cancers, but their applications in treating OSCC are limited. Alantolactone (ALT) possesses anticancer properties and also regulates mitochondrial events. In this study, we explored the effects of ALT on OSCC and the related mechanisms. METHODS: The OSCC cells were treated with varying concentrations and duration of ALT and N-Acetyl-L-cysteine (NAC). The cell viability and colony formation were assessed. The apoptotic rate was evaluated by flow cytometry with Annexin V-FITC/PI double staining. We used DCFH-DA and flow cytometry to detect reactive oxygen species (ROS) production and DAF-FM DA to investigate reactive nitrogen species (RNS) level. Mitochondrial function was reflected by mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP levels. KEGG enrichment analyses determined the mitochondrial-related hub genes involved in OSCC progression. Dynamin-related protein 1 (Drp1) overexpression plasmids were further transfected into the cells to analyze the role of Drp1 in OSCC progression. Immunohistochemistry staining and western blot verified the expression of the protein. RESULTS: ALT exerted anti-proliferative and pro-apoptosis effects on OSCC cells. Mechanistically, ALT elicited cell injury by promoting ROS production, mitochondrial membrane depolarization, and ATP depletion, which were reversed by NAC. Bioinformatics analysis showed that Drp1 played a crucial role in OSCC progression. OSCC patients with low Drp1 expression had a higher survival rate. The OSCC cancer tissues presented higher phosphorylated-Drp1 and Drp1 levels than the normal tissues. The results further showed that ALT suppressed Drp1 phosphorylation in OSCC cells. Moreover, Drp1 overexpression abolished the reduced Drp1 phosphorylation by ALT and promoted the cell viability of ALT-treated cells. Drp1 overexpression also reversed the mitochondrial dysfunction induced by ALT, with decreased ROS production, and increased mitochondrial membrane potential and ATP level. CONCLUSIONS: ALT inhibited proliferation and promoted apoptosis of oral squamous cell carcinoma cells via impairment of mitochondrial homeostasis and regulation of Drp1. The results provide a solid basis for ALT as a therapeutic candidate for treating OSCC, with Drp1 being a novel therapeutic target in treating OSCC.

The Drp1-Mediated Mitochondrial Fission Protein Interactome as an Emerging Core Player in Mitochondrial Dynamics and Cardiovascular Disease Therapy.

Mitochondria, the membrane-bound cell organelles that supply most of the energy needed for cell function, are highly regulated, dynamic organelles bearing the ability to alter both form and functionality rapidly to maintain normal physiological events and challenge stress to the cell. This amazingly vibrant movement and distribution of mitochondria within cells is controlled by the highly coordinated interplay between mitochondrial dynamic processes and fission and fusion events, as well as mitochondrial quality-control processes, mainly mitochondrial autophagy (also known as mitophagy). Fusion connects and unites neighboring depolarized mitochondria to derive a healthy and distinct mitochondrion. In contrast, fission segregates damaged mitochondria from intact and healthy counterparts and is followed by selective clearance of the damaged mitochondria via mitochondrial specific autophagy, i.e., mitophagy. Hence, the mitochondrial processes encompass all coordinated events of fusion, fission, mitophagy, and biogenesis for sustaining mitochondrial homeostasis. Accumulated evidence strongly suggests that mitochondrial impairment has already emerged as a core player in the pathogenesis, progression, and development of various human diseases, including cardiovascular ailments, the leading causes of death globally, which take an estimated 17.9 million lives each year. The crucial factor governing the fission process is the recruitment of dynamin-related protein 1 (Drp1), a GTPase that regulates mitochondrial fission, from the cytosol to the outer mitochondrial membrane in a guanosine triphosphate (GTP)-dependent manner, where it is oligomerized and self-assembles into spiral structures. In this review, we first aim to describe the structural elements, functionality, and regulatory mechanisms of the key mitochondrial fission protein, Drp1, and other mitochondrial fission adaptor proteins, including mitochondrial fission 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics 49 (Mid49), and mitochondrial dynamics 51 (Mid51). The core area of the review focuses on the recent advances in understanding the role of the Drp1-mediated mitochondrial fission adaptor protein interactome to unravel the missing links of mitochondrial fission events. Lastly, we discuss the promising mitochondria-targeted therapeutic approaches that involve fission, as well as current evidence on Drp1-mediated fission protein interactions and their critical roles in the pathogeneses of cardiovascular diseases (CVDs).

Acquired disorders of mitochondrial metabolism and dynamics in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is an orphan disease of the cardiopulmonary unit that reflects an obstructive pulmonary vasculopathy and presents with hypertrophy, inflammation, fibrosis, and ultimately failure of the right ventricle (RVF). Despite treatment using pulmonary hypertension (PH)-targeted therapies, persistent functional impairment reduces the quality of life for people with PAH and death from RVF occurs in approximately 40% of patients within 5 years of diagnosis. PH-targeted therapeutics are primarily vasodilators and none, alone or in combination, are curative. This highlights a need to therapeutically explore molecular targets in other pathways that are involved in the pathogenesis of PAH. Several candidate pathways in PAH involve acquired mitochondrial dysfunction. These mitochondrial disorders include: 1) a shift in metabolism related to increased expression of pyruvate dehydrogenase kinase and pyruvate kinase, which together increase uncoupled glycolysis (Warburg metabolism); 2) disruption of oxygen-sensing related to increased expression of hypoxia-inducible factor 1α, resulting in a state of pseudohypoxia; 3) altered mitochondrial calcium homeostasis related to impaired function of the mitochondrial calcium uniporter complex, which elevates cytosolic calcium and reduces intramitochondrial calcium; and 4) abnormal mitochondrial dynamics related to increased expression of dynamin-related protein 1 and its binding partners, such as mitochondrial dynamics proteins of 49 kDa and 51 kDa, and depressed expression of mitofusin 2, resulting in increased mitotic fission. These acquired mitochondrial abnormalities increase proliferation and impair apoptosis in most pulmonary vascular cells (including endothelial cells, smooth muscle cells and fibroblasts). In the RV, Warburg metabolism and induction of glutaminolysis impairs bioenergetics and promotes hypokinesis, hypertrophy, and fibrosis. This review will explore our current knowledge of the causes and consequences of disordered mitochondrial function in PAH.

Inhibition of diabetes-induced Drp1 deSUMOylation prevents retinal vascular lesions associated with diabetic retinopathy.

Retinal microvascular endothelial cell (RMEC) injury plays an important role in the pathophysiology diabetic retinopathy (DR). The GTPase dynamin-related protein 1 (Drp1), crucial to mitochondrial dynamics, has been implicated in hyperglycaemia-induced microvascular damage. Moreover, Drp1 can be deSUMOylated by the enzyme sentrin/SUMO-specific protease 3 (SENP3). Whether SENP3/deSUMOylated Drp1 can aggravate DR is unclear. Therefore, we designed this experiment to investigate the role of SENP3/desumoylated Drp1 in DR in vitro and in vivo. Murine RMECs (mRMECs) were classified into a control (CON), high-glucose (HG) and high-glucose + SENP3-siRNA (HG-siRNA) groups. The SENP3 and SUMOylated/deSUMOylated drp1 levels, mitochondrial morphology, mitochondrial membrane potential (MMP) and apoptosis rate were evaluated. In vivo, mice were assigned to a normal, type 2 diabetic or type 2 diabetic SENP3-siRNA mouse groups. Then, blood-retinal barrier function and retinal tissue structure were evaluated. As compared to those in the control group, the SENP3 and Drp1 levels, degree of mitochondrial fragmentation, extent of MMP loss and apoptosis rate of mRMECs were significantly increased in the HG group. However, inhibited SENP3 expression increased the level of SUMOylated Drp1 in the mRMECs and reduced the hyperglycaemia-induced mitochondrial damage and apoptosis rate. These experimental results were confirmed by diabetic animal experiments showing that inhibited SENP3 expression attenuated the increase in retinal permeability and diabetic retinopathy, suggesting that SENP3/deSUMOylated Drp1 activation aggravated DR by disrupting mitochondrial dynamics and apoptosis. Furthermore, blocking SENP3 expression significantly attenuated RMEC damage and DR.

PGAM5 regulates DRP1-mediated mitochondrial fission/mitophagy flux in lipid overload-induced renal tubular epithelial cell necroptosis.

The pathophysiology of renal lipid toxicity caused by excess adiposity is not well-understood. Necroptosis, a regulated form of cell death, is involved in injuring renal tubular epithelial cells (RTECs). Phosphoglycerate mutase 5 (PGAM5) is a key downstream effector of necroptosis. This study investigated the underlying mechanism of PGAM5 in promoting lipid-induced necroptosis in RTECs. HK2 cells (an immortalized proximal tubule epithelial cell line) were exposed to oleic acid (OA) to mimic the lipid overload environment in vitro. We found that OA suppressed HK2 cell proliferation, triggered cytoskeleton rupture and cell death. In OA-treated cells, upregulated expression of necroptosis pathway proteins, phosphorylated receptor-interacting protein-1/3 (pRIPK1/3), phosphorylated mixed lineage kinase domain-like protein (pMLKL), PGAM5, phosphorylated dynamin-related protein 1 (pDRP1S616), and downregulated pDRP1S637 expression were observed. This was accompanied by mitochondrial dysfunction (mitochondrial ROS overproduction and decreased mitochondrial membrane potential) and increased cellular necrosis, as reflected by Annexin V/ Propidium Iodide (PI) labeling. OA also induced the accumulation of LC3II and P62, blocking autophagosome fusion with lysosomes. Knockdown of PGAM5 could prevent these OA-induced changes. We propose inhibition of PGAM5 protects lipid-induced RTECs from necroptosis by reducing DRP1-mediated mitochondrial fission and improving mitophagy flux.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia.

RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

[Protective effect of edaravone on chlorpyrifos-induced brain injury in rats and its mechanism].

Objective: To investigate the protective effect of edaravone on chlorpyrifos-induced neuronal apoptosis and its mitochondrial mechanism. Methods: Under the principle of randomization and double-blindness, the rats were divided into control group, chlorpyrifos group, and edaravone group (n=6). The rats in edaravone group were treated with edaravone (10 mg/1.6 ml/kg, ip.) 1 h after chlorpyrifos injection. After continuous injection of chlorpyrifos and edaravone for 28 days, the learning and memory abilities of the rats were tested by open field and water maze tests. The rat brain tissue was collected after cardiac perfusion, and the neuronal damage in the hippocampus of the brain was detected by HE staining and the mitochondrial and nuclear damage were observed by transmission electron microscopy. The contents of Na+-K+-ATPase and ATP were measured to evaluate mitochondrial damage. The expression of mitochondrial fission protein DRP1 and phosphorylation at Ser 637 of DRP1 were determined by immunohistochemistry and immunoblotting. Results: Compared with the control group, the total movement distance and average speed of the rats in the chlorpyrifos group were decreased significantly within 3 minutes of the open field test (P＜0.01), and the escape latency within 1 minute of the water maze test was prolonged significantly. The number of platform crossings was reduced significantly (P＜0.01), the activity of ATPase in brain tissue was decreased significantly (P＜0.01) , the content of ATP and the phosphorylation level of Ser637 of mitochondrial DRP1 were decreased significantly (P＜0.05, P＜0.01). After edaravone treatment, the total movement distance and average speed of rats in the open field test were increased (P＜0.05), the latency in the water maze test was decreased, and the number of crossing platforms was increased (P＜0.01), brain pathological sections showed that nerve cells were arranged neatly, nucleus and mitochondrial damage was significantly improved, the activity of ATPase in brain tissue was increased (P＜0.01), the levels of ATP and mitochondrial DRP1 Ser637 phosphorylation increased (P＜0.05, P＜0.01).Conclusion: Edaravone alleviates chlorpyrifos-induced brain injury in rats by promoting the phosphorylation of DRP1 at Ser637.

Loss of PTEN-Induced Kinase 1 Regulates Oncogenic Ras-Driven Tumor Growth By Inhibiting Mitochondrial Fission.

Mitochondrial metabolism and dynamics (fission and fusion) critically regulate cell survival and proliferation, and abnormalities in these pathways are implicated in both neurodegenerative disorders and cancer. Mitochondrial fission is necessary for the growth of mutant Ras-dependent tumors. Here, we investigated whether loss of PTEN-induced kinase 1 (PINK1) - a mitochondrial kinase linked to recessive familial Parkinsonism - affects the growth of oncogenic Ras-induced tumor growth in vitro and in vivo. We show that RasG12D-transformed embryonic fibroblasts (MEFs) from PINK1-deficient mice display reduced growth in soft agar and in nude mice, as well as increased necrosis and decreased cell cycle progression, compared to RasG12D-transformed MEFs derived from wildtype mice. PINK1 re-expression (overexpression) at least partially rescues these phenotypes. Neither PINK1 deletion nor PINK1 overexpression altered Ras expression levels. Intriguingly, PINK1-deficient Ras-transformed MEFs exhibited elongated mitochondria and altered DRP1 phosphorylation, a key event in regulating mitochondrial fission. Inhibition of DRP1 diminished PINK1-regulated mitochondria morphological changes and tumor growth suggesting that PINK1 deficiency primarily inhibits Ras-driven tumor growth through disturbances in mitochondrial fission and associated cell necrosis and cell cycle defects. Moreover, we substantiate the requirement of PINK1 for optimal growth of Ras-transformed cells by showing that human HCT116 colon carcinoma cells (carrying an endogenous RasG13D mutation) with CRISPR/Cas9-introduced PINK1 gene deletions also show reduced mitochondrial fission and decreased growth. Our results support the importance of mitochondrial function and dynamics in regulating the growth of Ras-dependent tumor cells and provide insight into possible mechanisms underlying the lower incidence of cancers in Parkinson's disease and other neurodegenerative disorders.

Regulating mitochondrial homeostasis and inhibiting inflammatory responses through Celastrol.

Background: The high morbidity and mortality rate of coronary heart disease poses a serious threat to human health. Atherosclerosis, a chronic inflammation of the blood vessel wall, is a significant pathological process leading to coronary heart disease. Macrophage inflammation plays a crucial role in the occurrence and development of atherosclerosis. Methods: Macrophage inflammation model was constructed by lipopolysaccharide (LPS), and macrophages were treated with Celastrol at different concentrations (0, 0.1, 1, 10, 100 ng/mL) and different time points (0, 1, 3, 6, 12 h). Real-time quantitative PCR (qPCR) and Western Blot were used to detect the expression of Nur77 mRNA and protein. Macrophages were then pretreated with 100 nmol/L tripterine for 40min and co-cultured with 100 ng/mL LPS. The expression levels of inflammatory factors and chemokines, phosphorylation of phospho-dynamin-related protein 1 (p-Drp1) at Ser637 and expression of mitochondrial fusion protein mitochondrial fusion protein mitofusin-2 (Mfn2) were detected by qPCR, Western blot and ELISA, respectively. The changes of mitochondrial membrane potential were detected by JC-1 probe. Results: 100 nmol/L Celastrol can significantly inhibit LPS-induced inflammatory responses and down-regulate the expression levels of cytokines such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), tumor necrosis factor-α (TNF-α), chemokines (CCL-2, and CXCL-10), as well as chemokines. And Celastrol could regulate mitochondrial fission and fusion by promoting the phosphorylation of the Drp1 at the Ser637 site, thereby inhibiting mitochondrial fission. At the same time, by up-regulating the level of the Mfn2, Celastrol also promoted mitochondrial fusion. In addition, we found that the nuclear factor-k-gene binding (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 signaling pathways aided the drug's anti-inflammatory effects. We also explored the relationship between Celastrol and the nuclear receptor Nur77 and found that it could up-regulate the expression of Nur77. Conclusions: Our study found that Celastrol could reduce inflammation by regulating Drp1 dependent mitochondrial fission and fusion, as well as the ERK1/2, p38, NF-κB signaling pathways. This finding provides a strong direction for the development of new anti-inflammatory drugs for atherosclerosis.

Myostatin is involved in skeletal muscle dysfunction in chronic obstructive pulmonary disease via Drp-1 mediated abnormal mitochondrial division.

Background: Skeletal muscle dysfunction (SMD) is one of the most prominent extrapulmonary effects of chronic obstructive pulmonary disease (COPD). Myostatin negatively regulates the growth of skeletal muscle. We confirmed that myostatin expression is significantly increased in the quadriceps femoris muscle tissue of rats with COPD and is involved in the development of SMD in COPD, but the mechanism by which this occurs has yet to be uncovered. Dynamin-related protein 1 (Drp-1) has been shown to promote apoptosis and affect cellular energy metabolism by mediating enhanced mitochondrial division. Preliminary findings from our group illustrated that mitochondrial division and Drp-1 expression were increased in COPD quadriceps femoris cells. However, it is not yet clear whether mitochondrial dynamics are affected by myostatin in COPD quadriceps myocytes. Methods: The study sought to explore the effects and potential mechanisms of myostatin on skeletal muscle atrophy, mitochondrial dynamics, apoptosis, and the links between related processes in COPD. Results: Our findings showed that cigarette smoke exposure stimulated an increase in myostatin, increased superoxide production, decreased mitochondrial membrane potential, significantly promoted Drp-1-mediated mitochondrial fission, and promoted apoptosis. Conclusions: In summary, our study demonstrated that cigarette smoke led to increased Drp-1 expression and enhanced mitochondrial division by upregulating myostatin, which in turn promoted apoptosis and affected cellular energy metabolism, leading to the development of SMD in COPD. This study extends understandings of skeletal muscle function in COPD and provides a basis for the use of myostatin and Drp-1 as novel therapeutic targets for SMD in COPD.

Dipsacoside B Exerts a Beneficial Effect on Brain Injury in the Ischemic Stroke Rat through Inhibition of Mitochondrial E3 Ubiquitin Ligase 1.

BACKGROUND: Upregulation of mitochondrial E3 ubiquitin ligase 1 (Mul1) contributes to brain injury in ischemic stroke due to disturbance of mitochondrial dynamics, and bioinformatics analysis predicts that Mul1 is a potential target of Dipsacoside B. OBJECTIVE: The aim of the study was to explore whether Dipsacoside B can exert a beneficial effect on brain injury in the ischemic stroke rat via targeting Mul1. METHODS: The SD rat brains or PC12 cells were subjected to 2 h-ischemia or 8 h-hypoxia plus 24 h-reperfusion or 24 h-reoxygenation to establish the ischemic stroke rat model in vivo or in vitro, which were treated with Dipsacoside B at different dosages. The brain or PC12 cell injury, relevant protein levels and mitochondrial functions were measured by methods of biochemistry, flow cytometry or Western blot. RESULTS: The neurological dysfunction and brain injury (such as infarction and apoptosis) observed in the ischemic stroke rats were accompanied by increases in Mul1 and Dynamin-related protein 1 (Drp1) levels along with decreases in mitofusin 2 (Mfn2) level and ATP production. These effects were attenuated by Dipsacoside B. Consistently, cell injury (necroptosis and apoptosis) occurred in the PC12 cells exposed to hypoxia concomitant with the upregulation of Mul1 and Drp1 along with downregulation of Mfn2 and mitochondrial functions (such as increases in reactive oxygen species production and mitochondrial fission and decreases in mitochondrial membrane potential and ATP production).These phenomena were reversed in the presence of Dipsacoside B. CONCLUSION: Dipsacoside B can protect the rat brain against ischemic injury via inhibition of Mul1 due to the improvement of mitochondrial function.

Adiponectin receptor agonist AdipoRon blocks skin inflamm-ageing by regulating mitochondrial dynamics.

INTRODUCTION: Skin is susceptible to senescence-associated secretory phenotype (SASP) and inflamm-ageing partly owing to the degeneration of mitochondria. AdipoRon (AR) has protective effects on mitochondria in metabolic diseases such as diabetes. We explored the role of AR on mitochondria damage induced by skin inflamm-ageing and its underlying mechanism. METHODS: Western blot, immunofluorescence and TUNEL staining were used to detect inflammatory factors and apoptosis during skin ageing. Transmission electron microscopy, ATP determination kit, CellLight Mitochondria GFP (Mito-GFP), mitochondrial stress test, MitoSOX and JC-1 staining were used to detect mitochondrial changes. Western blot was applied to explore the underlying mechanism. Flow cytometry, scratch test, Sulforhodamine B assay and wound healing test were used to detect the effects of AR on cell apoptosis, migration and proliferation. RESULTS: AR attenuated inflammatory factors and apoptosis that increased in aged skin, and improved mitochondrial morphology and function. This process at least partly depended on the suppression of dynamin-related protein 1 (Drp1)-mediated excessive mitochondrial division. More specifically, AR up-regulated the phosphorylation of Drp1 at Serine 637 by activating AMP-activated protein kinase (AMPK), thereby inhibiting the mitochondrial translocation of Drp1. Moreover, AR reduced mitochondrial fragmentation and the production of superoxide, preserved the membrane potential and permeability of mitochondria and accelerated wound healing in aged skin. CONCLUSION: AR rescues the mitochondria in aged skin by suppressing its excessive division mediated by Drp1.

BORIS/CTCFL expression activates the TGFß signaling cascade and induces Drp1 mediated mitochondrial fission in neuroblastoma.

The cancer-testis antigen CTCFL/BORIS (Brother of Regulator of Imprinted Sites) also known, as a paralog of CTCF -the "master weaver of the genome" is a key transcriptional regulator. Both CTCF and BORIS can bind to the same promoter sequence and recruit diverse proteins. BORIS is also known to be associated with actively translating ribosomes suggesting new roles of BORIS in gene expression. Various studies have attempted to elucidate the role of BORIS in different cell types for the development of targeted therapy depending on molecular signatures and genetic aberrations associated with the disease type. The current study is focused on its role in neuroblastoma. Here, we have deciphered the role of BORIS on TGFß1 pathway which is highly affected by embryonic CTCFL expression. BORIS stabilized the SMAD3 and SMAD4 transcripts leading to prolonged TGFß activation. Further, loss of BORIS abrogated both the canonical and non-canonical TGFß signaling suggesting the dependency of TGFß on BORIS. The effect on the metabolic profile of the neuroblastoma cells were analyzed with change in BORIS expression levels. Also, ectopic expression of BORIS leads to Drp1 phosphorylation (Ser616) enhancing mitochondrial fission followed by a switch in cellular metabolism towards glycolysis. This cellular metabolism switch was in turn supported with a reduction in oxygen consumption rate upon BORIS expression. Interestingly methylome analysis revealed patterns of global histone methylation, a mechanism that regulate important signaling pathways in neuroblastoma. This study analyzes the consequence of BORIS expression in neuroblastoma cells and thereby elucidate its downstream targets, which could help in designing effective therapeutic for treating neuroblastoma. Similar results were obtained in both MYCN amplified and non-MYCN neuroblastoma cell lines, indicating a common mechanism of BORIS/CTCFL action in neuroblastoma.