3-(2-4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3h)-quinazolinone and Disease-Models--Animal

The current therapeutic strategy for the management of acute myocardial infarction (AMI) is to return blood flow into the occluded coronary artery of the heart, a process defined as reperfusion. However, reperfusion itself can increase mortality rates in AMI patients because of cardiac tissue damage and dysfunction, which is termed 'ischaemia/reperfusion (I/R) injury'. Mitochondria play an important role in myocardial I/R injury as disturbance of mitochondrial dynamics, especially excessive mitochondrial fission, is a predominant cause of cardiac dysfunction. Therefore, pharmacological intervention and therapeutic strategies which modulate the mitochondrial dynamics balance during I/R injury could exert great beneficial effects to the I/R heart. This review comprehensively summarizes and discusses the effects of mitochondrial fission inhibitors as well as mitochondrial fusion promoters on cardiac and mitochondrial function during myocardial I/R injury. The comparison of the effects of both compounds given at different time-points during the course of I/R injury (i.e. prior to ischaemia, during ischaemia and at the reperfusion period) are also summarized and discussed. Finally, this review also details important information which may contribute to clinical practices using these drugs to improve the quality of life in AMI patients.

Topics: Animals; Cardiotonic Agents; Cell Line; Disease Models, Animal; GTP Phosphohydrolases; Humans; Hydrazones; Mitochondria, Heart; Mitochondrial Dynamics; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Peptide Fragments; Quinazolinones; Tacrolimus; Ventricular Function, Left

Allergic rhinitis (AR) is a common allergic disorder, afflicting thousands of human beings. Aberrant mitochondrial dynamics are important pathological elements for various immune cell dysfunctions and allergic diseases. However, the connection between mitochondrial dynamics and AR remains poorly understood. This study aimed to determine whether mitochondrial dynamics influence the inflammatory response in AR.. In the present study, we established a murine model of AR by sensitization with ovalbumin (OVA). Then, we investigated the mitochondrial morphology in mice with AR by transmission electron microscopy and confocal fluorescence microscopy, and evaluated the role of Mdivi-1 (an inhibitor of mitochondrial fission) on allergic symptoms, inflammatory responses, allergic-related signals, and reactive oxygen species formation.. There was a notable enhancement in mitochondrial fragmentation in the nasal mucosa of mice following OVA stimulation, whereas Mdivi-1 prevented aberrant mitochondrial morphology. Indeed, Mdivi-1 alleviated the rubbing and sneezing responses in OVA-sensitized mice. Compared with vehicle-treated ones, mice treated with Mdivi-1 exhibited a reduction in interleukin (IL)-4, IL-5, and specific IgE levels in both serum and nasal lavage fluid, and shown an amelioration in inflammatory response of nasal mucosa. Meanwhile, Mdivi-1 treatment was associated with a suppression in JAK2 and STAT6 activation and reactive oxygen species generation, which act as important signaling for allergic response.. Our findings reveal mitochondrial dynamics modulate the allergic responses in AR. Mitochondrial dynamics may represent a promising target for the treatment of AR.

Topics: Animals; Disease Models, Animal; Humans; Immunoglobulin E; Inflammation; Mice; Mitochondrial Dynamics; Reactive Oxygen Species; Rhinitis, Allergic

Angiotensin II (AngII) is a potential contributor to the development of abdominal aortic aneurysm (AAA). In aortic vascular smooth muscle cells (VSMCs), exposure to AngII induces mitochondrial fission via dynamin-related protein 1 (Drp1). However, pathophysiological relevance of mitochondrial morphology in AngII-associated AAA remains unexplored. Here, we tested the hypothesis that mitochondrial fission is involved in the development of AAA.. Immunohistochemistry was performed on human AAA samples and revealed enhanced expression of Drp1. In C57BL6 mice treated with AngII plus β-aminopropionitrile, AAA tissue also showed an increase in Drp1 expression. A mitochondrial fission inhibitor, mdivi1, attenuated AAA size, associated aortic pathology, Drp1 protein induction, and mitochondrial fission but not hypertension in these mice. Moreover, western-blot analysis showed that induction of matrix metalloproteinase-2, which precedes the development of AAA, was blocked by mdivi1. Mdivi1 also reduced the development of AAA in apolipoprotein E-deficient mice infused with AngII. As with mdivi1, Drp1+/- mice treated with AngII plus β-aminopropionitrile showed a decrease in AAA compared to control Drp1+/+ mice. In abdominal aortic VSMCs, AngII induced phosphorylation of Drp1 and mitochondrial fission, the latter of which was attenuated with Drp1 silencing as well as mdivi1. AngII also induced vascular cell adhesion molecule-1 expression and enhanced leucocyte adhesion and mitochondrial oxygen consumption in smooth muscle cells, which were attenuated with mdivi1.. These data indicate that Drp1 and mitochondrial fission play salient roles in AAA development, which likely involves mitochondrial dysfunction and inflammatory activation of VSMCs.

Topics: Aminopropionitrile; Angiotensin II; Animals; Anti-Inflammatory Agents; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Case-Control Studies; Cell Adhesion; Cells, Cultured; Disease Models, Animal; Dynamins; Humans; Leukocytes; Male; Mice, Inbred C57BL; Mice, Knockout, ApoE; Mitochondria, Muscle; Mitochondrial Dynamics; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oxygen Consumption; Phosphorylation; Quinazolinones

Tyrosine kinase Fyn is a member of the Src kinase family, which is involved in neuroinflammation, apoptosis, and oxidative stress. Its role in intracerebral hemorrhage (ICH) is not fully understood. In this study, we found that Fyn was significantly elevated in human brain tissue after ICH. Accordingly, we investigated the role of Fyn in a rat ICH model, which was constructed by injecting blood into the right basal ganglia. In this model, Fyn expression was significantly upregulated in brain tissue adjacent to the hematoma. SiRNA-induced Fyn knockdown was neuroprotective for secondary cerebral damage, as demonstrated by reduced brain edema, suppression of the modified neurological severity score, and mitigation of blood-brain barrier permeability and neuronal damage. Fyn downregulation reduced apoptosis following ICH, as indicated by downregulation of apoptosis-related proteins AIF, Cyt.c, caspase 3, and Bax; upregulation of anti-apoptosis-related protein Bcl-2; and decreased tunnel staining. Mdivi-1, a Drp1 inhibitor, reversed Fyn overexpression induced pro-apoptosis. However, Fyn did not significantly affect inflammation-related proteins NF-κB, TNF-α, caspase 1, MPO, IL-1β, or IL-18 after ICH. Fyn activated Drp1 signaling by phosphorylating Drp1 at serine 616, which increased apoptosis after ICH in rats. This study clarifies the relationship between Fyn, apoptosis, and inflammation following ICH and provides a new strategy for exploring the prevention and treatment of ICH. KEY MESSAGES: ICH induced an increase in Fyn expression in human and rat cerebral tissues. Knockdown of Fyn prevented cerebral damage following ICH. Inhibition of Fyn had no significant effects on inflammatory responses. However, the downregulation of Fyn exerted neuroprotective effects on apoptosis. Fyn perturbed ICH-induced cell apoptosis by interacting with and phosphorylating (Ser616) Drp1 in a rat ICH model.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Blood-Brain Barrier; Brain; Brain Edema; Cerebral Hemorrhage; Disease Models, Animal; Down-Regulation; Dynamins; Gene Knockdown Techniques; Humans; Male; Nerve Tissue Proteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-fyn; Quinazolinones; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Signal Transduction; Specific Pathogen-Free Organisms

Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis.. We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages.. We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

Topics: Animals; Atherosclerosis; Disease Models, Animal; Dynamins; Endoplasmic Reticulum; Hep G2 Cells; Humans; Liver; Mice, Knockout, ApoE; Mitochondria, Liver; Mitochondrial Dynamics; PCSK9 Inhibitors; Proprotein Convertase 9; Proteasome Endopeptidase Complex; Protein Interaction Maps; Proteolysis; Proteostasis; Quinazolinones; Secretory Pathway

Intracerebral hemorrhage (ICH) is a serious public health problem with high rates of death and disability. The neuroprotective effect of Growth Differentiation Factor 11 (GDF11) in ICH has been initially proved by our previous study. Oxidative stress (OS) plays crucial roles in mediating subsequent damage of ICH. However, whether and how mitochondrial dynamic events and function participated in ICH pathophysiology, and how mitochondrial function and OS interreacted in the neuroprotective process of GDF11 in ICH remains unclarified. Based on the rat model of ICH and in vitro cell model, we demonstrated that GDF11 could alleviate ICH induced neurological deficits, brain edema, OS status, neuronal apoptosis and inflammatory reaction. In addition, mitochondrial functional and structural impairments were obviously restored by GDF11. Treatment with antioxidant protected against erythrocyte homogenate (EH) induced cell injury by restoring OS status and mitochondrial fusion fission imbalance, which was similar to the effect of GDF11 treatment. Further, inhibition of mitochondrial division with Mdivi-1 attenuated mitochondrial functional defects and neuronal damages. In conclusion, our results for the first time proposed that GDF11 protected the post-ICH secondary injury by suppressing the feedback loop between mitochondrial ROS production and mitochondrial dynamic alteration, resulting in attenuated mitochondrial function and amelioration of neural damage.

Topics: Animals; Antioxidants; Apoptosis; Brain Injuries; Cerebral Hemorrhage; Disease Models, Animal; Enzyme Activation; Growth Differentiation Factors; Humans; Inflammation; L-Lactate Dehydrogenase; Male; Mitochondrial Dynamics; Neurons; Neuroprotective Agents; Oxidative Stress; Quinazolinones; Rats; Reactive Oxygen Species

Atopic dermatitis (AD) is a chronic inflammatory cutaneous disorder with few treatment options. Dynamin-related protein 1 (Drp1)-dependent mitochondrial fission contributes to the activation of NLRP3 inflammasome, and inhibiting Drp1 has been become an attractive therapeutic strategy for inflammatory diseases. This study aimed to investigate the effects of Drp1 inhibitor mdivi-1 on experimental AD. We firstly detected the effects of mdivi-1 on primary human keratinocytes in an inflammatory cocktail-induced AD-related inflammation in vitro. Results showed that mdivi-1 inhibited NLRP3 inflammasome activation and pyroptosis which were evidenced by decreased expression of NLRP3, ASC, cleavage of caspase-1, GSDMD-NT, mature interleukin (IL)-1β and IL-18 in keratinocytes under AD-like inflammation. Next, mouse model of AD-like skin lesions was induced by epicutaneous application of 2,4-dinitrochlorobenzene (DNCB) and mdivi-1 (25 mg/kg/day, days 5-33 during construction of AD model) was intraperitoneally injected into DNCB-induced mice. AD mice with mdivi-1 treatment exhibited ameliorated AD symptoms, lower serum IgE level, and reduced epidermal thickening, mast cells infiltration, and production of IL-4, IL-5 and IL-13 in the lesional tissues. Indeed, mdivi-1 significantly inhibited NLRP3 inflammasome activation and pyroptotic injury occurred in DNCB-treated skin tissues. Mechanically, mdivi-1 regulated the expression of mitochondrial dynamic proteins and suppressed the activation of NF-κB signal pathway which is an upstream of NLRP3 inflammasome both in vitro and in vivo. This study demonstrated that mdivi-1 could protect against experimental AD through inhibiting the activation of NLRP3 inflammasome and subsequent inflammatory cytokine release, and mdivi-1 might exert this function by inhibiting mitochondrial fission and subsequently blocking NF-κB pathway.

Topics: Administration, Topical; Animals; Dermatitis, Atopic; Dinitrochlorobenzene; Disease Models, Animal; Dynamins; Female; Humans; Inflammasomes; Keratinocytes; Mice; Mice, Inbred BALB C; NLR Family, Pyrin Domain-Containing 3 Protein; Quinazolinones

Mitochondria play an essential role in energy metabolism. Oxygen deprivation can poison cells and generate a chain reaction due to the free radical release. In patients with sepsis, the kidneys tend to be the organ primarily affected and the proximal renal tubules are highly susceptible to energy metabolism imbalances. Dynamin-related protein 1 (DRP1) is an essential regulator of mitochondrial fission. Few studies have confirmed the role and mechanism of DRP1 in acute kidney injury (AKI) caused by sepsis. We established animal and cell sepsis-induced AKI (S-AKI) models to keep DRP1 expression high. We found that Mdivi-1, a DRP1 inhibitor, can reduce the activation of the NOD-like receptor pyrin domain-3 (NLRP3) inflammasome-mediated pyroptosis pathway and improve mitochondrial function. Both S-AKI models showed that Mdivi-1 was able to prevent the mitochondrial content release and decrease the expression of NLRP3 inflammasome-related proteins. In addition, silencing NLRP3 gene expression further emphasized the pyroptosis importance in S-AKI occurrence. Our results indicate that the possible mechanism of action of Mdivi-1 is to inhibit mitochondrial fission and protect mitochondrial function, thereby reducing pyroptosis. These data can provide a potential theoretical basis for Mdivi-1 potential use in the S-AKI prevention.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cell Line; Disease Models, Animal; Down-Regulation; Dynamins; Inflammasomes; Kidney Tubules; Lipopolysaccharides; Male; Mice, Inbred C57BL; Mitochondria; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Quinazolinones; RNA, Small Interfering; Sepsis

Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and triggers NLRP3 inflammasome activation. FK866 (a NAMPT inhibitor) exerts a neuroprotective effect in ischemia/reperfusion injury through the suppression of mitochondrial dysfunction. We explored the effects of FK866 on pyroptosis and inflammation mediated by Drp1 in a cardiac arrest/cardiopulmonary resuscitation (CA/CPR) rat model.. Healthy male Sprague-Dawley rats were subjected to 7 min CA by trans-esophageal electrical stimulation followed by CPR. The surviving rats were treated with FK866 (a selective inhibitor of NAMPT), Mdivi-1 (Drp1 inhibitor), FK866 + Mdivi-1, or vehicle and then underwent 24 h reperfusion. Hematoxylin and eosin staining and immunohistochemistry (to detect NSE) were used to evaluate brain injury. We performed immunofluorescent staining to analyze NLRP3 and GSDMD expression in microglia or astrocytes and western blot to determine expression of NLRP3, IL-1β, GSDMD, Drp1, and Mfn2. Transmission electron microscopy was used to observe mitochondria.. FK866 significantly decreased pathological damage to brain tissue, inhibited the activation of NLRP3 in microglia or astrocytes, downregulated the expression of NLRP3, IL-1β, GSDMD, p-Drp1 protein, upregulated Mfn2 and improve mitochondrial morphology.. Our results demonstrated that FK866 protects the brain against ischemia-reperfusion injury in rats after CA/CPR by inhibiting pyroptosis and inflammation mediated by Drp1.

Topics: Acrylamides; Animals; Anti-Inflammatory Agents; Brain; Cardiopulmonary Resuscitation; Disease Models, Animal; Dynamins; Inflammasomes; Inflammation Mediators; Male; Mitochondria; Neuroprotective Agents; NLR Family, Pyrin Domain-Containing 3 Protein; Piperidines; Pyroptosis; Quinazolinones; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction

Pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) dysfunction. However, the underlying mechanisms of PASMC dysfunction remain largely unknown. Here, we show that mitochondrial fragmentation contributes to PASMC dysfunction through enhancement of endoplasmic reticulum (ER) stress. PASMC dysfunction accompanied by mitochondrial fragmentation and ER stress was observed in the pulmonary arteries of hypoxia-induced rats with PAH, as well as isolated PASMCs under hypoxia. Treatment with Mdivi-1 inhibited mitochondrial fragmentation and ER stress and improved PASMC function in isolated PASMCs under hypoxia, while Drp1 overexpression increased mitochondrial fragmentation and ER stress, impairing PASMC function in isolated PASMCs under normoxia. However, inhibition of ER stress using ER stress inhibitors showed a negligible effect on mitochondrial morphology but improved PASMC function during hypoxia. Additionally, we found that mitochondrial fragmentation-promoted ER stress was dependent on mitochondrial reactive oxygen species. Furthermore, inhibition of mitochondrial fragmentation using Mdivi-1 attenuated mitochondrial fragmentation and ER stress in hypoxic PASMCs and improved the pulmonary artery smooth muscle function in hypoxic rats. These results suggest that hypoxia induces pulmonary artery smooth muscle dysfunction through mitochondrial fragmentation-mediated ER stress and that mitochondrial morphology is a potential target for treatment of hypoxia-induced pulmonary artery smooth muscle dysfunction.

Topics: Animals; Cell Hypoxia; Cells, Cultured; Disease Models, Animal; Dynamins; Endoplasmic Reticulum Stress; Hypoxia; Male; Mitochondria, Muscle; Mitochondrial Dynamics; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pulmonary Arterial Hypertension; Pulmonary Artery; Quinazolinones; Rats, Sprague-Dawley; Reactive Oxygen Species

Increasing evidence has implicated dysfunctional mitochondria in the pathophysiology of neurodegenerative disorders. Selective degradation of dysfunctional mitochondria has been termed mitophagy and constitutes a pivotal component of mitochondrial quality control to maintain cellular homeostasis. Mitochondrial fission plays a prominent role in controlling mitochondrial shape and function. However, it is unclear whether mitochondrial fission in the context of eliminating damaged mitochondria is involved in traumatic brain injury (TBI). We examined the role of mitochondrial division inhibitor 1 (Mdivi1), a small-molecule inhibitor of dynamin-related protein (Drp1), in general autophagy and mitophagy after controlled cortical impact (CCI).. Mitophagy and the role of Drp1 in this process after CCI were examined using Western blotting, electron microscopy, double immunofluorescence staining, neurological severity scores, and hematoxylin and eosin staining. Statistical analysis was performed using 1-way analysis of variance, followed by the least significant difference test or the Games-Howell test.. The rats exposed to CCI exhibited induction of mitophagy and fragmentation of mitochondria. When fission was blocked with Mdivi1, the mitochondria became excessively long and interconnected. Inhibition of Drp1 blocked the induction of mitophagy specifically, which aggravated neurological manifestations and neuronal apoptosis. Mdivi1 activated caspase-3 and caspase-9, implying that selective degradation of damaged mitochondria by autophagy markedly decreased cell apoptosis induced by TBI and, thus, promoted cell survival.. The findings from the present study support the hypothesis that Drp1-dependent mitochondrial fission contributes to mitophagy in TBI, and further understanding of the regulatory mechanisms of Drp1 will provide opportunities to develop novel strategies against TBI.

Topics: Animals; Autophagy; Brain Injuries; Cell Death; Cerebral Cortex; Disease Models, Animal; Dynamins; Male; Mitochondria; Mitophagy; Quinazolinones; Rats; Rats, Sprague-Dawley

The regulation of intracellular mitochondria degradation is mediated by mitophagy. While studies have shown that mitophagy can lead to mitochondrial dysfunction and cell damage, the role of Mdivi-1 and mitophagy remains unclear in acute lung injury (ALI) pathogenesis. In this study, we demonstrated that Mdivi-1, which is widely used as an inhibitor of mitophagy, ameliorated acute lung injury assessed by HE staining, pulmonary microvascular permeability assay, measurement of wet/dry weight (W/D) ratio, and oxygenation index (PaO2/FiO2) analysis. Then, the mitophagy related proteins were evaluated by western blot. The results indicated that LPS-induced activation of mitophagy was inhibited by Mdivi-1 treatment. In addition, we found that Mdivi-1 protected A549 cells against LPS-induced mitochondrial dysfunction. We also found that Mdivi-1 reduced pulmonary cell apoptosis in the LPS-challenged rats and protected pulmonary tissues from oxidative stress (represented by the content of superoxide dismutase, malondialdehyde and lipid peroxides in lung). Moreover, Mdivi-1 treatment ameliorated LPS-induced lung inflammatory response and cells recruitment. These findings indicate that Mdivi-1 mitigates LPS-induced apoptosis, oxidative stress, and inflammation in ALI, which may be associated with mitophagy inhibition. Thus, the inhibition of mitophagy may represent a potential therapy for treating ALI.

Topics: A549 Cells; Acute Lung Injury; Animals; Apoptosis; Disease Models, Animal; Humans; Lipopolysaccharides; Male; Mitochondria; Mitophagy; Oxidative Stress; Quinazolinones; Rats; Rats, Sprague-Dawley

Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth, however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to its oncogenicity, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a two-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared to vehicle. We found that the chief tumor suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.

Topics: Animals; Carcinoma, Pancreatic Ductal; CRISPR-Cas Systems; Disease Models, Animal; Dynamins; Enzyme Inhibitors; GTP Phosphohydrolases; Leflunomide; Mice; Mice, Knockout; Mitochondria; Mitochondrial Dynamics; Mitophagy; Oxidative Phosphorylation; Pancreatic Neoplasms; Quinazolinones; Survival Rate

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. Our previous studies proved that the inhibition of Drp1 may help attenuate traumatic brain injury (TBI)-induced functional outcome and cell death through maintaining normal mitochondrial morphology and inhibiting activation of apoptosis. However, the molecular mechanisms of Drp1 after TBI remain poorly understood. In this study, we investigated the role of mitochondrial division inhibitor 1 (Mdivi-1), a small molecule inhibitor of Drp1, in underlying mechanisms of general autophagy and mitochondria autophagy (mitophagy) after experimental TBI. In vivo, we found that autophagosomes accumulated in cortical neurons at 24h after TBI, owing to the enhanced autophagy indicated by the accumulation of LC3 and the decrease of p62; but Mdivi-1 reversed the enhancement. Mdivi-1 also alleviated the number of LC3 puncta and TUNEL-positive structures in cells, indicating that autophagy maybe involved in Mdivi-1's anti-apoptosis effects. Then, the expression level of mitochondrial dynamics related and mitophagy related proteins was assessed using the isolated mitochondria. The results showed that TBI-induced mitochondrial fission (represented by Drp1), mtDNA concentration down-regulation and PTEN induced putative kinase 1 (PINK1)-Parkin mediated mitophagy activation were all inhibited by Mdivi-1. In addition, TBI-induced blood-brain barrier (BBB) disruption and matrix metalloproteinases (MMP)-9 expression up-regulation were inhibited following Mdivi-1 treatment. In vitro, Mdivi-1 significantly alleviated the scratch injury-induced cell death, loss of mitochondrial membrane potential, reactive oxygen species (ROS) production and ATP reduction in primary cortical neurons (PCNs). Additionally, the lysosome inhibitor chloroquine (CQ) abrogated the Mdivi-1-induced decrease in autophagosomes accumulation and cell death at 24h both in the basal state and under the conditions of scratch cell injury. Together, these data demonstrate that Mdivi-1 mitigates TBI-induced BBB disruption and cell death at least in part by a mechanism involving inhibiting autophagy dysfunction and mitophagy activation.

Topics: Animals; Apoptosis; Autophagosomes; Autophagy; Biomarkers; Blood-Brain Barrier; Brain Injuries, Traumatic; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Dynamins; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Male; Membrane Transport Modulators; Mice, Inbred Strains; Mitophagy; Nerve Tissue Proteins; Neurons; Quinazolinones; Random Allocation

Dynamin-related protein 1 (Drp1) regulates mitochondrial fission, it has been proven that inhibition of Drp1 by mdivi-1 improves survival and attenuates cerebral ischemic injury after cardiac arrest. In this study, we compared the effects of Drp1 inhibition with therapeutic hypothermia on post-resuscitation neurologic injury in a rat model of cardiac arrest. Rats were randomized into 4 groups: mdivi-1 treatment group (n = 39), hypothermic group (n = 38), normothermic group (n = 41), and sham group (n = 12). The rats in the mdivi-1 treatment group were received intravenously 1.2 mg/kg of mdivi-1 at 1 minute after the return of spontaneous circulation (ROSC). In rats in hypothermia group, rapid cooling was initiated at 5 minutes after resuscitation, and the core temperature was maintained to 33 ± 0.5°C for 2 hours. The results showed that both Drp1 inhibition and therapeutic hypothermia increased 3-day survival time (all P <0.05) and improved neurologic function up to 72 hours post cardiac arrest. In addition, both Drp1 inhibition and therapeutic hypothermia decreased cell injury, apoptosis in hippocampal cornu ammonis 1 region and brain mitochondrial dysfunction including adenosine triphosphate production, reactive oxygen species and mitochondrial membrane potential after cardiac arrest. Moreover, therapeutic hypothermia decreased mitochondrial Drp1 expression and mitochondrial fission after cardiac arrest. In conclusion, inhibition of Drp1 has a similar effect to therapeutic hypothermia on neurologic outcome after resuscitation in this cardiac arrest rat model, and the neuroprotective effects of therapeutic hypothermia are associated with inhibition of mitochondrial fission.

Topics: Animals; Disease Models, Animal; Dynamins; Heart Arrest; Hypothermia, Induced; Male; Mitochondrial Dynamics; Neuroprotective Agents; Quinazolinones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Leukemia is a type of hematopoietic stem cell malignant cloned disease with high mortality. Cisplatin-based chemotherapy is one of the most common treatments for leukemia. Similar to other chemotherapeutic agents, cisplatin resistance has become a serious issue in cancer therapy. In the present study, we investigated the role of mitochondrial dynamics in the antineoplastic activity of cisplatin in murine leukemia L1210 cells. Firstly, the L1210 cell line resistant to cisplatin (L1210/DDP) was established. Compared to its parental cell line, the IC50 value of cisplatin in the L1210/DDP cells was increased 10-fold. Mitofusins (Mfn1 and Mfn2), mitochondrial outer membrane fusion proteins, were markedly upregulated in the L1210/DDP cells, whereas the expression of fission protein Drp1 and inner membrane fusion protein OPA1 were not significantly altered. In addition, mitofusins were also upregulated in the parental L1210 cells subjected to cisplatin stress. To investigate the role of mitochondrial dynamics in the antineoplastic activity of cisplatin, the effect of mitochondrial division inhibitor (Mdivi)-1 on cisplatin‑induced cell death, caspase-3 cleavage and ROS production was examined in L1210 cells. We found that 5 µM of Mdivi-1 efficiently attenuated cisplatin-induced cell death, caspase activation and intracellular ROS increase in L1210 cells. Our data indicated that mitochondrial dynamics play an important role in the antineoplastic activity of cisplatin, and mitofusin-mediated mitochondrial fusion may be involved in the process of cisplatin resistance in leukemia cells. Therefore, the present study revealed that mitochondrial dynamics may be a potential target used to improve the antineoplastic activity of cisplatin in leukemia in the future.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase 3; Cisplatin; Disease Models, Animal; Drug Resistance, Neoplasm; Humans; Leukemia; Leukemia L1210; Mice; Mitochondrial Dynamics; Quinazolinones

Functional and structural damages to mitochondria have been critically associated with the pathogenesis of Down syndrome (DS), a human multifactorial disease caused by trisomy of chromosome 21 and associated with neurodevelopmental delay, intellectual disability and early neurodegeneration. Recently, we demonstrated in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice -a widely used model of DS - a severe impairment of mitochondrial bioenergetics and biogenesis and reduced NPC proliferation. Here we further investigated the origin of mitochondrial dysfunction in DS and explored a possible mechanistic link among alteration of mitochondrial dynamics, mitochondrial dysfunctions and defective neurogenesis in DS. We first analyzed mitochondrial network and structure by both confocal and transmission electron microscopy as well as by evaluating the levels of key proteins involved in the fission and fusion machinery. We found a fragmentation of mitochondria due to an increase in mitochondrial fission associated with an up-regulation of dynamin-related protein 1 (Drp1), and a decrease in mitochondrial fusion associated with a down-regulation of mitofusin 2 (Mnf2) and increased proteolysis of optic atrophy 1 (Opa1). Next, using the well-known neuroprotective agent mitochondrial division inhibitor 1 (Mdivi-1), we assessed whether the inhibition of mitochondrial fission might reverse alteration of mitochondrial dynamics and mitochondrial dysfunctions in DS neural progenitors cells. We demonstrate here for the first time, that Mdivi-1 restores mitochondrial network organization, mitochondrial energy production and ultimately improves proliferation and neuronal differentiation of NPCs. This research paves the way for the discovery of new therapeutic tools in managing some DS-associated clinical manifestations.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Down Syndrome; Dynamins; Energy Metabolism; GTP Phosphohydrolases; Hippocampus; Mice; Mitochondria; Mitochondrial Dynamics; Neurogenesis; Optic Atrophy, Autosomal Dominant; Quinazolinones

Mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer's disease, which likely plays a critical role in the pathogenesis of disease. Increasing evidence suggests abnormal mitochondrial dynamics as important underlying mechanisms. In this study, we characterized marked mitochondrial fragmentation and abnormal mitochondrial distribution in the pyramidal neurons along with mitochondrial dysfunction in the brain of Alzheimer's disease mouse model CRND8 as early as 3 months of age before the accumulation of amyloid pathology. To establish the pathogenic significance of these abnormalities, we inhibited mitochondrial fragmentation by the treatment of mitochondrial division inhibitor 1 (mdivi-1), a mitochondrial fission inhibitor. Mdivi-1 treatment could rescue both mitochondrial fragmentation and distribution deficits and improve mitochondrial function in the CRND8 neurons both in vitro and in vivo. More importantly, the amelioration of mitochondrial dynamic deficits by mdivi-1 treatment markedly decreased extracellular amyloid deposition and Aβ1-42/Aβ1-40 ratio, prevented the development of cognitive deficits in Y-maze test and improved synaptic parameters. Our findings support the notion that abnormal mitochondrial dynamics plays an early and causal role in mitochondrial dysfunction and Alzheimer's disease-related pathological and cognitive impairments in vivo and indicate the potential value of restoration of mitochondrial dynamics as an innovative therapeutic strategy for Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidogenic Proteins; Animals; Brain; Cognition Disorders; Disease Models, Animal; Mice; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Neurons; Pyramidal Cells; Quinazolinones

Mitochondria play an important role in many cellular and physiologic functions. Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis. The most common complaint of human immunodeficiency virus (HIV)-sensory neuropathy is pain on the soles in patients with HIV, but the exact molecular mechanisms of HIV neuropathic pain are not clear. In the present study, we investigated the role of mitochondrial dynamin-related protein 1 (Drp1, a GTPase that mediates mitochondrial fission) in the perineural HIV coat glycoprotein gp120-induced neuropathic pain state.. Neuropathic pain was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. Mechanical threshold was tested using von Frey filaments. The mechanical threshold response was assessed over time using the area under curves. Intrathecal administration of antisense oligodeoxynucleotide (ODN) against Drp1, mitochondrial division inhibitor-1 (mdivi-1), or phenyl-N-tert-butylnitrone (a reactive oxygen species scavenger) was given. The expression of spinal Drp1 was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging.. Intrathecal administration of either antisense ODN against Drp1 or mdivi-1 decreased mechanical allodynia (a sensation of pain evoked by nonpainful stimuli) in the gp120 model. Intrathecal ODN or mdivi-1 did not change basic mechanical threshold in sham surgery rats. Intrathecal Drp1 antisense ODN decreased the spinal expression of increased Drp1 protein induced by peripheral gp120 application. Intrathecal phenyl-N-tert-butylnitrone reduced mechanical allodynia. Furthermore, both intrathecal Drp1 antisense ODN and mdivi-1 reversed the upregulation of mitochondrial superoxide in the spinal dorsal horn in the gp120 neuropathic pain state.. These data suggest that mitochondrial division plays a substantial role in the HIV gp120-related neuropathic pain state through mitochondrial reactive oxygen species and provides evidence for a novel approach to treating chronic pain in patients with HIV.

Topics: Analgesics; Animals; Cyclic N-Oxides; Disease Models, Animal; Dynamins; Free Radical Scavengers; HIV Envelope Protein gp120; HIV Infections; Hyperalgesia; Injections, Spinal; Male; Mitochondria; Mitochondrial Dynamics; Oligonucleotides, Antisense; Pain Threshold; Posterior Horn Cells; Quinazolinones; Rats, Sprague-Dawley; Recombinant Proteins; Sciatica; Superoxides; Time Factors

Mitochondria dysfunction, an enormous potential crisis, has attracted increasing attention. Disturbed regulation of mitochondrial dynamics, the balance of mitochondrial fusion and fission, has been implicated in neurodegenerative diseases, such as Parkinson׳s disease and cerebral ischemia/reperfusion. However the role of mitochondrial dynamics in traumatic brain injury (TBI) has not been illuminated. The aim of the present study was to investigate the role of Mdivi-1, a small molecule inhibitor of a key mitochondrial fission protein dynamin-related protein 1 (Drp1), in TBI-induced cell death and functional outcome deficits. Protein expression of Drp1 was first investigated. Outcome parameters consist of motor test, Morris water maze, brain edema and lesion volume. Cell death was detected by propidium iodide (PI) labeling, and mitochondrial morphology was assessed using transmission electron microscopy. In addition, the expression of apoptosis-related proteins cytochrome c (cyt-c) and caspase-3 was investigated. Our findings showed that up-regulation of Drp1 expression started at 1h post-TBI and peaked at 24 h, but inhibition of Drp1 by Mdivi-1 significantly alleviated TBI-induced behavioral deficits and brain edema, reduced morphological change of mitochondria, and decreased TBI-induced cell death together with lesion volume. Moreover, treatment with Mdivi-1 remarkably inhibited TBI-induced the release of cyt-c from mitochondria to cytoplasm, and activation of caspase-3 at 24 h after TBI. Taken together, these data imply that inhibition of Drp1 may help attenuate TBI-induced functional outcome and cell death through maintaining normal mitochondrial morphology and inhibiting activation of apoptosis.

Topics: Animals; Brain; Brain Edema; Brain Injuries; Caspase 3; Cell Death; Cytochromes c; Disease Models, Animal; Dynamins; Male; Maze Learning; Mice, Inbred ICR; Mitochondria; Motor Activity; Neuroprotective Agents; Quinazolinones; Random Allocation; Recovery of Function

Kainic acid (KA)-induced excitotoxicity promotes cytoplasmic calcium accumulation, oxidative stress, and apoptotic signaling, leading to hippocampal neuronal death. Mitochondria play a critical role in neuroinflammation and the oxidative stress response. Mitochondrial morphology is disrupted during KA-induced seizures; however, it is not clear whether mitochondrial fission or fusion factors are involved in KA-induced neuronal death.. We investigated the effect of Mdivi-1, a chemical inhibitor of the mitochondrial fission protein Drp1, on mitochondrial morphology and function in KA-injected mice. Mdivi-1 pretreatment significantly reduced seizure activity and increased survival rates of KA-treated mice. Mdivi-1 was protective against mitochondrial morphological disruption, and it reduced levels of phosphorylated Drp1 (Ser616) and Parkin recruitment to mitochondria. By contrast, levels of mitochondrial fusion factors did not change. Mdivi-1 also reduced KA-induced neuroinflammation and glial activation.. We conclude that inhibition of mitochondrial fission attenuates Parkin-mediated mitochondrial degradation and protects from KA-induced hippocampal neuronal cell death.

Topics: Animals; Calcium-Binding Proteins; Cell Death; Cyclooxygenase 2; Disease Models, Animal; Dynamins; Hippocampus; HSP72 Heat-Shock Proteins; Kainic Acid; Male; Mice, Inbred ICR; Microfilament Proteins; Mitochondria; Mitochondrial Dynamics; Mitophagy; Neuroglia; Neuroimmunomodulation; Neurons; Neuroprotective Agents; Quinazolinones; Random Allocation; Seizures; Survival Analysis; Ubiquitin-Protein Ligases

When given prior to brain ischemia, mitochondrial division inhibitor-1 (mdivi-1) attenuates the brain damage caused by ischemia. Here, we investigated the potential effects of post-ischemia mdivi-1 treatment (1mg/kg, i.p., administered immediately after 2h of ischemia and prior to reperfusion) using a MCAO rat model. Mdivi-1 treatment decreased infarct volume and improved neurological function. In addition, cytochrome C release was attenuated, and neuronal apoptosis was decreased. The mitochondrial fission protein dynamin-related protein 1 (Drp1) was decreased in the mitochondrial fraction but increased in the cytosolic fraction. Mdivi-1 treatment augmented the increases in the mRNA expression of peroxisome proliferator-activated receptor coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcriptional factor A. In conclusion, when given after ischemia and prior to reperfusion, mdivi-1 can protect against brain damage by inhibiting the mitochondria-mediated apoptosis induced by mitochondrial fission. Post-ischemia mdivi-1 treatment might promote I/R-induced mitochondrial biogenesis.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cytochromes c; Disease Models, Animal; Dynamins; Male; Mitochondria; Mitochondrial Dynamics; Neurons; Neuroprotective Agents; Organelle Biogenesis; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury

Prolonged seizure activity may result in mitochondrial dysfunction and lead to cell death in the hippocampus. Mitochondrial fission may occur in an early stage of neuronal cell death. This study examined the role of the mitochondrial fission protein dynamin-related protein 1 (Drp1) in the hippocampus following status epilepticus.. Kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 area in Sprague Dawley rats to induce prolonged seizure activity. Biochemical analysis, electron microscopy, and immunofluorescence staining were performed to evaluate the subsequent molecular and cellular events. The effects of pretreatment with a mitochondrial fission protein inhibitor, Mdivi-1 (2 nmol), were also evaluated.. Phosphorylation of Drp1 at serine 616 (p-Drp1(Ser616)) was elevated from 1 to 24 h after the elicited seizure activity. Pretreatment with Mdivi-1 decreased the Drp1 phosphorylation at Ser616 and limited the mitochondrial fission. Mdivi-1 rescued the Complex I dysfunction, decreased the levels of oxidized proteins, decreased the activation of cytochrome c/caspase-3 signaling, and blunted cell death in CA3 neurons.. Our findings suggest that activation of p-Drp1(Ser616) is related to seizure-induced neuronal damage. Modulation of p-Drp1(Ser616) expression is accompanied by decreases in mitochondrial fission, mitochondrial dysfunction, and oxidation, providing a neuroprotective effect against seizure-induced hippocampal neuronal damage.

Topics: Animals; Apoptosis; Caspase 3; Disease Models, Animal; Dynamins; Excitatory Amino Acid Agonists; Functional Laterality; Gene Expression Regulation; Hippocampus; Kainic Acid; Male; Mitochondrial Dynamics; NAD; Neurons; Phosphopyruvate Hydratase; Quinazolinones; Rats; Rats, Sprague-Dawley; Serine; Status Epilepticus

Survival following sudden cardiac arrest is poor despite advances in cardiopulmonary resuscitation and the use of therapeutic hypothermia. Dynamin-related protein 1, a regulator of mitochondrial fission, is an important determinant of reactive oxygen species generation, myocardial necrosis, and left ventricular function following ischemia/reperfusion injury, but its role in cardiac arrest is unknown. We hypothesized that dynamin-related protein 1 inhibition would improve survival, cardiac hemodynamics, and mitochondrial function in an in vivo model of cardiac arrest.. Laboratory investigation.. University laboratory.. Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent an 8-minute KCl-induced cardiac arrest followed by 90 seconds of cardiopulmonary resuscitation. Mice were then blindly randomized to a single IV injection of Mdivi-1 (0.24 mg/kg), a small molecule dynamin-related protein 1 inhibitor or vehicle (dimethyl sulfoxide).. Following resuscitation from cardiac arrest, mitochondrial fission was evidenced by dynamin-related protein 1 translocation to the mitochondrial membrane and a decrease in mitochondrial size. Mitochondrial fission was associated with increased lactate and evidence of oxidative damage. Mdivi-1 administration during cardiopulmonary resuscitation inhibited dynamin-related protein 1 activation, preserved mitochondrial morphology, and decreased oxidative damage. Mdivi-1 also reduced the time to return of spontaneous circulation (116 ± 4 vs 143 ± 7 s; p < 0.001) during cardiopulmonary resuscitation and enhanced myocardial performance post-return of spontaneous circulation. These improvements were associated with significant increases in survival (65% vs 33%) and improved neurological scores up to 72 hours post cardiac arrest.. Post-cardiac arrest inhibition of dynamin-related protein 1 improves time to return of spontaneous circulation and myocardial hemodynamics, resulting in improved survival and neurological outcomes in a murine model of cardiac arrest. Pharmacological targeting of mitochondrial fission may be a promising therapy for cardiac arrest.

Topics: Aconitate Hydratase; Animals; Disease Models, Animal; Dynamins; Female; Heart Arrest; Immunoblotting; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Mitochondrial Dynamics; Quinazolinones; Random Allocation

Glaucoma is the leading cause of irreversible blindness and is characterized by slow and progressive degeneration of the optic nerve head axons and retinal ganglion cell (RGC), leading to loss of visual function. Although oxidative stress and/or alteration of mitochondrial (mt) dynamics induced by elevated intraocular pressure (IOP) are associated with this neurodegenerative disease, the mechanisms that regulate mt dysfunction-mediated glaucomatous neurodegeneration are poorly understood. Using a mouse model of glaucoma, DBA/2J (D2), which spontaneously develops elevated IOP, as well as an in vitro RGC culture system, we show here that oxidative stress, as evidenced by increasing superoxide dismutase 2 (SOD2) and mt transcription factor A (Tfam) protein expression, triggers mt fission and loss by increasing dynamin-related protein 1 (DRP1) in the retina of glaucomatous D2 mice as well as in cultured RGCs exposed to elevated hydrostatic pressure in vitro. DRP1 inhibition by overexpressing DRP1 K38A mutant blocks mt fission and triggers a subsequent reduction of oxidative stress, as evidenced by decreasing SOD2 and Tfam protein expression. DRP1 inhibition promotes RGC survival by increasing phosphorylation of Bad at serine 112 in the retina and preserves RGC axons by maintaining mt integrity in the glial lamina of glaucomatous D2 mice. These findings demonstrate an important vicious cycle involved in glaucomatous neurodegeneration that starts with elevated IOP producing oxidative stress; the oxidative stress then leads to mt fission and a specific form of mt dysfunction that generates further oxidative stress, thus perpetuating the cycle. Our findings suggest that DRP1 is a potential therapeutic target for ameliorating oxidative stress-mediated mt fission and dysfunction in RGC and its axons during glaucomatous neurodegeneration. Thus, DRP1 inhibition may provide a new therapeutic strategy for protecting both RGCs and their axons in glaucoma and other optic neuropathies.

Topics: Animals; Axons; bcl-Associated Death Protein; Disease Models, Animal; DNA-Binding Proteins; Dynamins; Female; Gene Expression Regulation; Glaucoma; GTP Phosphohydrolases; High Mobility Group Proteins; Humans; Intraocular Pressure; Mice; Mice, Inbred DBA; Mitochondrial Dynamics; Mutation; Optic Disk; Peptide Fragments; Phosphorylation; Protective Agents; Quinazolinones; Retinal Ganglion Cells; Signal Transduction; Superoxide Dismutase; Tissue Culture Techniques

Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has been reported to be involved in mitochondria apoptosis pathways. However, the role of Drp1 in a rat model of cardiac arrest remains unknown. In this study, we found that activation of Drp1 in the mitochondria was increased after cardiac arrest and inhibition of Drp1 by 1.2 mg/kg of mitochondrial division inhibitor-1 (Mdivi-1) administration after the restoration of spontaneous circulation (ROSC) significantly protected against cerebral ischemic injury, shown by the increased 72-h survival rate and improved neurological function. Moreover, the increase of the vital neuron and the reduction of cytochrome c (CytC) release, apoptosis-inducing factor (AIF) translocation and caspase-3 activation in the brain indicate that this protection might result from the suppression of neuron apoptosis. Altogether, these results indicated that Drp1 is activated after cardiac arrest and the inhibition of Drp1 is protective against cerebral ischemic injury in a rat of cardiac arrest model via inhibition of the mitochondrial apoptosis pathway.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Dynamins; Heart Arrest; Male; Mitochondria; Neurons; Neuroprotective Agents; Quinazolinones; Random Allocation; Rats, Sprague-Dawley

Disturbance of the balance between mitochondrial fission and fusion has been implicated in cerebral ischemia and several neurodegenerative diseases, whereas the underlying mechanisms remain poorly understood. In the present study, we attempted to investigate the role of dynamin-related protein 1 (Drp1), a key mitochondrial fission protein, in the pathogenesis of cerebral ischemia.. Using Drp1 siRNA or Mdivi-1, a small molecule inhibitor of Drp1, we examined the effect of Drp1 knockdown or inhibition on oxygen-glucose deprivation (OGD)-induced mitochondrial dysfunction and death of SH-SY-5Y cells. Cell death and viability were evaluated with LDH and MTT assays, respectively, and mitochondrial morphology, mitochondrial membrane potential (Δψm), and ATP production were assessed using epifluorescence microscopy, flow cytometry, and HPLC, respectively. Moreover, to examine the effect of Drp1 inhibition on ischemic brain injury, middle cerebral artery occlusion (MCAO) mice were injected (i.p.) with Mdivi1, and blood-brain barrier permeability, brain water content, and cell apoptosis were assessed.. Knockdown or inhibition of Drp1 by Mdivi-1 significantly attenuated OGD-induced cell death in SH-SY-5Y cells, associated with reduced morphological change of mitochondria and attenuated Bax insertion,oligomerization. Moreover, treatment of the MCAO mice with Mdivi-1 remarkably reduced the infarct volume and neurological deficits in a dose-dependent manner, associated with marked reduction of mitochondrial fragmentation and BAX expression.. Down-regulation or inhibition of Drp1 may reduce cerebral ischemic damage through maintaining normal mitochondrial morphology and function, and decreasing Bax insertion and oligomerization in mitochondria.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blood-Brain Barrier; Brain Edema; Capillary Permeability; Cell Hypoxia; Cell Line, Tumor; Disease Models, Animal; Gene Expression Regulation; Glucose; Humans; Infarction, Middle Cerebral Artery; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Neuroblastoma; Quinazolinones

Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known.. We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1.. These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

Topics: Animals; Arterioles; Blood Platelets; Disease Models, Animal; Dynamins; Exocytosis; Fibrinolytic Agents; GTP Phosphohydrolases; Humans; Lasers; Membrane Fusion; Mice; Microtubule-Associated Proteins; Mitochondrial Proteins; P-Selectin; Quinazolinones; Rabbits; Secretory Vesicles; Serotonin; Thrombosis; Time Factors; Vascular System Injuries

Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction caused, in part, by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs; however, their relationship and relevance to the development of PAH are unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate serine 616, causes mitochondrial fission. It is, however, unknown whether mitochondrial fission is a prerequisite for proliferation.. We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential.. Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1-dependent phosphorylation of DRP1 at serine 616. In normal PASMCs, HIF-1α activation by CoCl(2) or desferrioxamine causes DRP1-mediated fission. HIF-1α inhibition reduces DRP1 activation, prevents fission, and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMCs and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function, and hemodynamics in experimental PAH.. DRP-1-mediated mitotic fission is a cell-cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

Topics: Animals; Antihypertensive Agents; Case-Control Studies; CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Cobalt; Cyclin B1; Disease Models, Animal; Dynamins; Enzyme Activation; Familial Primary Pulmonary Hypertension; Genetic Therapy; Glycolysis; GTP Phosphohydrolases; Humans; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Microtubule-Associated Proteins; Mitochondria, Muscle; Mitochondrial Proteins; Mitosis; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Pulmonary Artery; Quinazolinones; Rats; Rats, Sprague-Dawley; RNA Interference; Serine; Time Factors; Transfection

While oxidative stress has been implicated in small-fiber painful peripheral neuropathies, antioxidants are only partially effective to treat patients. We have tested the hypothesis that Drp1 (dynamin-related protein 1), a GTPase that catalyzes the process of mitochondrial fission, which is a mechanism central for the effect and production of reactive oxygen species (ROS), plays a central role in these neuropathic pain syndromes. Intrathecal administration of oligodeoxynucleotide antisense against Drp1 produced a decrease in its expression in peripheral nerve and markedly attenuated neuropathic mechanical hyperalgesia caused by HIV/AIDS antiretroviral [ddC (2',3'-dideoxycytidine)] and anticancer (oxaliplatin) chemotherapy in male Sprague Dawley rats. To confirm the role of Drp1 in these models of neuropathic pain, as well as to demonstrate its contribution at the site of sensory transduction, we injected a highly selective Drp1 inhibitor, mdivi-1, at the site of nociceptive testing on the dorsum of the rat's hindpaw. mdivi-1 attenuated both forms of neuropathic pain. To evaluate the role of Drp1 in hyperalgesia induced by ROS, we demonstrated that intradermal hydrogen peroxide produced dose-dependent hyperalgesia that was inhibited by mdivi-1. Finally, mechanical hyperalgesia induced by diverse pronociceptive mediators involved in inflammatory and neuropathic pain-tumor necrosis factor α, glial-derived neurotrophic factor, and nitric oxide-was also inhibited by mdivi-1. These studies provide support for a substantial role of mitochondrial fission in preclinical models of inflammatory and neuropathic pain.

Topics: Analysis of Variance; Animals; Anti-HIV Agents; Antineoplastic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Dynamins; Epinephrine; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Hydrogen Peroxide; Hyperalgesia; Male; Nerve Growth Factor; Neuralgia; Nitric Oxide Donors; Nitro Compounds; Oligodeoxyribonucleotides, Antisense; Organoplatinum Compounds; Oxaliplatin; Pain Measurement; Quinazolinones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha; Zalcitabine