Mfn2 Protein Research Articles

Effect and molecular mechanism of Sulforaphane alleviates brain damage caused by acute carbon monoxide poisoning:Network pharmacology analysis, molecular docking, and experimental evidence.

Sulforaphane (SFN) has attracted much attention due to its ability on antioxidant, anti-inflammatory, and anti-apoptotic properties, while its functional targets and underlying mechanism of action on brain injury caused by acute carbon monoxide poisoning (ACOP) have not been fully elucidated. Herein, we used a systematic network pharmacology approach to explore the mechanism of SFN in the treatment of brain damage after ACOP. In this study, the results of network pharmacology demonstrated that there were a total of 81 effective target genes of SFN and 36 drug-disease targets, which were strongly in connection with autophagy-animal signaling pathway, drug metabolism, and transcription disorders in cancer. Upon the further biological function and KEGG signaling pathway enrichment analysis, a large number of them were involved in neuronal death, reactive oxygen metabolic processes and immune functions. Moreover, based on the results of bioinformatics prediction associated with multiple potential targets and pathways, the AMP-activated protein kinase (AMPK) signaling pathway was selected to elucidate the molecular mechanism of SFN in the treatment of brain injury caused by ACOP. The following molecular docking analysis also confirmed that SFN can bind to AMPKα well through chemical bonds. In addition, an animal model of ACOP was established by exposure to carbon monoxide in a hyperbaric oxygen chamber to verify the predicted results of network pharmacology. We found that the mitochondrial ultrastructure of neurons in rats with ACOP was seriously damaged, and apoptotic cells increased significantly. The histopathological changes were obviously alleviated, apoptosis of cortical neurons was inhibited, and the number of Nissl bodies was increased in the SFN group as compared with the ACOP group (p < .05). Besides, the administration of SFN could increase the expressions of phosphorylated P-AMPK and MFN2 proteins and decrease the levels of DRP1, Caspase3, and Casapase9 proteins in the brain tissue of ACOP rats. These findings suggest that network pharmacology is a useful tool for traditional Chinese medicine (TCM) research, SFN can effectively inhibit apoptosis, protect cortical neurons from the toxicity of carbon monoxide through activating the AMPK pathway and may become a potential therapeutic strategy for brain injury after ACOP.

SIRT4 is a regulator of human skeletal muscle fatty acid metabolism influencing inner and outer mitochondrial membrane-mediated fusion

ObjectiveThe mitochondrial phenotype, governed by the balance of fusion-fission, is a key determinant of energy metabolism. The inner and outer mitochondrial membrane (IMM) fusion proteins optic atrophy 1 (OPA1) and Mitofusin 1 and 2 (Mfn1/2) play an important role in this process. Recent evidence also shows that Sirtuin 4 (SIRT4), located within the mitochondria, is involved in the regulation of fatty acid oxidation. The purpose of this study was to determine if SIRT4 expression regulates inner and outer mitochondrial-mediated fusion and substrate utilization within differentiated human skeletal muscle cells (HSkMC). Material and methodsSIRT4 expression was knocked down using small interfering RNA (siRNA) transfection in differentiated HSkMC. Following knockdown, mitochondrial respiration was determined by high-resolution respirometry (HRR) using the Oroboros Oxygraph O2k. Live cell confocal microscopy, quantified using the Mitochondrial Network Analysis (MiNA) toolset, was used to examine mitochondrial morphological change. This was further examined through the measurement of key metabolic and mitochondrial morphological regulators (mRNA and protein) induced by knockdown. ResultsSIRT4 knockdown resulted in a significant decrease in LEAK respiration, potentially explained by a decrease in ANT1 protein expression. Knockdown further increased oxidative phosphorylation and protein expression of key regulators of fatty acid metabolism. Quantitative analysis of live confocal imaging of fluorescently labelled mitochondria following SIRT4 knockdown supported the role SIRT4 plays in the regulation of mitochondrial morphology, as emphasized by an increase in mitochondrial network branches and junctions. Measurement of key regulators of mitochondrial dynamics illustrated a significant increase in mitochondrial fusion proteins Mfn1, OPA1 respectively, indicative of an increase in mitochondrial size. ConclusionsThis study provides evidence of a direct relationship between the mitochondrial phenotype and substrate oxidation in HSkMC. We identify SIRT4 as a key protagonist of energy metabolism via its regulation of IMM and OMM fusion proteins, OPA1 and Mfn1. SIRT4 knockdown increases mitochondrial capacity to oxidize fatty acids, decreasing LEAK respiration and further increasing mitochondrial elongation via its regulation of mitochondrial fusion.

The mitochondrial fusion-associated protein MFN2 can be used as a novel prognostic molecule for clear cell renal cell carcinoma

BackgroundMitofusin 2 (MFN2) plays an important role in many tumors, but how its role in renal clear cell carcinoma needs further research.MethodsIn this study, we analyzed the expression of MFN2 in renal clear cell carcinoma tissues and normal kidney tissues through the Cancer Genome Atlas (TCGA) database and our clinical samples.Enrichment analysis was performed to determine MFN2-related pathways and biological functions. The correlation of MFN2 expression with immune cells was analyzed.The correlation of the expression of methylation and the methylation sites of MFN2 were analyzed by UALCAN and TCGA databases. Univariate / multivariate COX risk regression and Kaplan-Meier methods were used to determine the prognostic value of MFN2.Nomograms were drawn to predict overall survival (OS) at 1,3, and 5 years. We investigated the role of MFN2 in renal cancer cells using CCK 8, clone formation, wound healing assay, and methylase qPCR experiments.ResultsMFN2 is poorly expressed in renal clear cell carcinoma compared to normal kidney tissue,and is significantly negatively associated with TNM stage, histological grade and pathological stage.MFN2 was directly associated with OS after multivariate Cox regression analysis.MFN2 shows a hypomethylation state and shows a positive correlation with multiple methylation sites.Signaling pathways through functional enrichment to B-cell receptors and oxidative stress-induced senescence.Moreover, the low expression of MFN2 was positively correlated with the degree of immune cell infiltration in a variety of immune cells.In vitro experiments showed that overexpression of MFN2 significantly inhibited the proliferation and migration of renal clear cells and promoted methylation.ConclusionsIn conclusion, MFN2 can be used as a novel prognostic marker for renal clear cell carcinoma and requires further investigation of its role in tumor development.

Bilberry anthocyanins attenuate mitochondrial dysfunction via β-catenin/TCF pathway in Alzheimer’s disease

Alzheimer’s disease (AD) is a common form of neurodegeneration. In this study, we investigated the modulating effect of bilberry anthocyanins (BAs) on AD-like pathology in the APP/PSEN1 transgenic mouse and then explored the underlying mechanisms using the HT22 cells. We found that BAs significantly reduced the amyloid plaque load and the levels of β-amyloid 1–42 (Aβ42) in the brain and serum of APP/PSEN1 mice. Furthermore, BAs improved mitochondrial morphology and reduced brain NO and ROS levels in the APP/PSEN1 mice. Additional experiments using immortalized HT22 cells showed that BAs protected against Aβ42-induced cytotoxicity by reducing ROS levels and restoring mitochondrial homeostasis, and these effects were partly mediated by upregulation of the mitochondrial homeostasis-related protein Mfn2 via promoting the interaction between β-catenin and TCF7 in nuclear. Together, the results show that BAs have a protective effect against the development of AD, partly through restoration of mitochondrial homeostasis in a β-catenin/TCF-dependent manner.

Near-Infrared BODIPY Photosensitizer for Modulating Mitochondrial Fusion Proteins and Inhibiting Choroidal Neovascularization.

Photosensitizers have emerged as cytotoxic reactive oxygen species (ROS) activators in photodynamic therapy (PDT), which induced cell apoptosis. As the major contributors to ROS and oxidative stress, mitochondria play an important role in cell apoptosis. Although there are many reports about near-infrared 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as photosensitizers (PSs) for PDT, this kind of PS has rarely been used for treating mitochondrial function and choroidal neovascularization application at the same time. Herein, a novel near-infrared PS (BDP2) characterized by good water solubility, long wavelength excitation, and high ROS quantum yield has been made. Under near-infrared light irradiation, BDP2 would generate ROS with high yield, induce a mitochondrial morphology change, and trigger cell apoptosis by changing the fusion protein level. Deep investigation revealed that BDP2 can cause oxidative stress, break the balance between fusion and fission of mitochondrial dynamics protein through decreasing fusion protein MFN2 and OPA1 expression, and finally cause cell apoptosis. Due to these characteristics, the BDP2 PS was used to treat choroidal neovascularization in animal models and can inhibit neovascularization.

Dexamethasone inhibits IL-8 via glycolysis and mitochondria-related pathway to regulate inflammatory pain

BackgroundDexamethasone (Dexa) has been recently found to exert an analgesic effect, whose action is closely related to IL-8. However, whether dexamethasone induces antinociception via glycolysis and mitochondria-related pathways is still unclear.MethodsRight hind paw inflammatory pain in mice was induced by intraplantar injection of Freund’s Complete Adjuvant (FCA). Von Frey test was then used to measure the paw withdrawal threshold. The detection of glycolysis and mitochondrial pathway-related proteins and IL-8 were determined by Western blot and ELISA. The potential interaction between Dexa and fructose-1,6-bisphosphate (FBP, a PKM2 activator) was examined by simulation predictions using molecular docking.ResultsIntrathecal administration of Dexa (20 µg/20 µL) had an obvious analgesic effect in FCA-treated mice, which was counteracted by the glycolysis inhibitor 2-deoxyglucose (2-DG, 5 mg/20 µL) or the mitochondria-related pathway inhibitor oligomycin complex (Oligo, 5 µg/20 µL). In the glycolysis pathway, Dexa decreased GLUT3 and had no impact on HIF-1α expression during FCA-induced inflammation. Additionally, Dexa further increased the PKM2 level, accompanied by the formation of hydrogen bonds between Dexa and the PKM2 activator fructose-1,6-bisphosphate (FBP). In the mitochondrial pathway, Dexa downregulated the expression of Mfn2 protein but not the PGC-1α and SIRT-1 levels in the spinal cord. Moreover, both 2-DG and Oligo decreased Mfn2 expression. Finally, IL-8 level was reduced by the single or combined administration of Dexa, 2-DG, and Oligo.ConclusionDexa attenuated IL-8 expression via glycolysis and mitochondrial pathway-related proteins, thus mediating the analgesic effect during inflammatory pain.

Down-regulation of the mitochondrial fusion protein Opa1/Mfn2 promotes cardiomyocyte hypertrophy in Su5416/hypoxia-induced pulmonary hypertension rats

BackgroundMaladaptive right ventricular (RV) remodeling is the most important pathological feature of pulmonary hypertension (PH), involving processes such as myocardial hypertrophy and fibrosis. A growing number of studies have shown that mitochondria-associated endoplasmic reticulum membranes (MAMs) are involved in various physiological and pathological processes, such as calcium homeostasis, lipid metabolism, inflammatory response, mitochondrial dynamics, and autophagy/mitophagy. The abnormal expression of MAMs-related factors is closely related to the occurrence and development of heart-related diseases. However, the role of MAM-related factors in the maladaptive RV remodeling of PH rats remains unclear. Methods and resultsWe first obtained the transcriptome data of RV tissues from PH rats induced by Su5416 combined with hypoxia treatment (SuHx) from the Gene Expression Omnibus (GEO) database. The results showed that two MAMs-related genes (Opa1 and Mfn2) were significantly down-regulated in RV tissues of SuHx rats, accompanied by significant up-regulation of cardiac hypertrophy-related genes (such as Nppb and Myh7). Subsequently, using the SuHx-induced PH rat model, we found that the downregulation of mitochondrial fusion proteins Opa1 and Mfn2 may be involved in maladaptive RV remodeling by accelerating mitochondrial dysfunction. Finally, at the cellular level, we found that overexpression of Opa1 and Mfn2 could inhibit hypoxia-induced mitochondrial fission and reduce ROS production in H9c2 cardiomyocytes, thereby retarded the progression of cardiomyocyte hypertrophy. ConclusionsThe down-regulation of mitochondrial fusion protein Opa1/Mfn2 can accelerate cardiomyocyte hypertrophy and then participate in maladaptive RV remodeling in SuHx-induced PH rats, which may be potential targets for preventing maladaptive RV remodeling.

Saponins from Panax japonicus improve neuronal mitochondrial injury of aging rats

Context Panax japonicus is the dried rhizome of Panax japonicus C.A. Mey. (Araliaceae). Saponins from Panax japonicus (SPJ) exhibit anti-oxidative and anti-aging effects. Objective We evaluated the neuroprotective effects of SPJ on aging rats. Materials and methods Sprague-Dawley rats (18-months-old) were randomly divided into aging and SPJ groups (n = 8). Five-month-old rats were taken as the adult control (n = 8). The rats were fed a normal chow diet or the SPJ-containing diet (10 or 30 mg/kg) for 4 months. An in vitro model was established by d-galactose (d-Gal) in the SH-SY5Y cell line and pretreated with SPJ (25 and 50 µg/mL). The neuroprotection of SPJ was evaluated via Nissl staining, flow cytometry, transmission electron microscopy and Western blotting in vivo and in vitro. Results SPJ improved the neuronal degeneration and mitochondrial morphology that are associated with aging. Meanwhile, SPJ up-regulated the protein levels of mitofusin 2 (Mfn2) and optic atrophy 1 (Opa1) and down-regulated the protein level of dynamin-like protein 1 (Drp1) in the hippocampus of aging rats (p < 0.05 or p < 0.01 vs. 22 M). The in vitro studies also demonstrated that SPJ attenuated d-Gal-induced cell senescence concomitant with the improvement in mitochondrial function; SPJ, also up-regulated the Mfn2 and Opa1 protein levels, whereas the Drp1 protein level (p < 0.05 or p < 0.01 vs. d-Gal group) was down-regulated. Discussion and conclusions Further research on the elderly population will contribute to the development and utilization of SPJ for the treatment of neurodegenerative disorders.

Abstract P202: Changes In Cerebral Mitochondrial Function In Intrauterine Growth-Restricted Offspring Before The Development Of Hypertension And Cerebrovascular Dysfunction

Background: Preeclampsia (PE), new-onset gestational hypertension (HTN) affects 3-8% of all births in the USA. PE is the leading cause for intrauterine growth restriction (IUGR). IUGR children have increased risks of HTN and cerebrovascular disease (CVD) later in life. Prior studies in our lab show adult male IUGR rat offspring (OS) to develop HTN, CVD, and cerebral mitochondrial dysfunction (mtDys) at 17 weeks while female IUGR OS exhibit CVD. The role of mtDys in HTN is unknown and is the focus of our study. We hypothesize that IUGR OS will have mtDys prior to HTN. Methods: Our surgical PE animal model utilized the reduced-uterine-perfusion pressure (RUPP) surgery at GD 14. OS from NP served as controls (CON) while OS from RUPP mothers were IUGR. At 12 weeks, OS BP was measured alongside cerebral mtDys through mitochondrial fusion and fission, electron-transport chain (ETC) proteins, and respiration. Results: BP did not change between sexes or among IUGR and CON offspring. Male IUGR OS showed elevated mtRes (State 2: 199 ± 1.08 v. 76 ± 1.13 pmol/mg, p &lt; .05; State 3: 555 ± 3.02 v. 245 ± 1.77 pmol/mg, p &lt; .05; State 4: 346 ± 1.66 v. 85 ± 1.12 pmol/mg, p &lt; .05). Female IUGR and CON OS had no changes in BP or mtRes. Male IUGR OS had increased mito-fusion proteins MFN-1 (176 ± 1.68 v. 100 ± .57 IU/protein/CON %, p &lt; .05), MFN-2 (155 ± 1.36 v. 100 ± .74 IU/protein/CON %, p &lt; .05) and OPA-1 (166 ± 1.99 v. 100 ± .73 IU/protein/CON %, p &lt; .05). DRP-1 had no changes in males; females had no changes in fusion or fission. Male IUGR OS had changes in ETC proteins (CI: 8 ± 9.34 v. 100 ± .82 IU/protein/CON %, p &lt; .05; CII: 23 ± .53 v. 100 ± .52 IU/protein/CON %, p &lt; .05; CIII: 26 ± .23 v. 100 ± .41 IU/protein/CON %, p &lt; .05), as did female IUGR OS (CI: 12 ± .24 v. 100 ± .86 IU/protein/CON %, p &lt; .05; CII: 29 ± .39 v. 100 ± .61 IU/protein/CON %, p &lt; .05; CIII: 39 ± .56 v. 100 ± .62 IU/protein/CON %, p &lt; .05; CIV: 76 ± .74 v. 100 ± .73 IU/protein/CON %, p &lt; .05) vs. CON OS. Summary: Male IUGR OS have elevated mtRes, changed ETC proteins, and fusion proteins that may lead to mtDys and HTN development. This study is clinically relevant because specific cerebral therapy for mtDys for IUGR OS may prevent HTN and CVD development. Future studies will examine changes in mtDys causing male HTN and protection from HTN in females.

Acetaldehyde dehydrogenase 2 ameliorates lung endothelial barrier and balances mitochondrial dynamics in mice with acute lung injury

To investigate the protective effects of acetaldehyde dehydrogenase 2 (ALDH2) against lipopolysaccharide (LPS)- induced acute lung injury (ALI) in mice and explore the possible mechanisms. Sixty C57BL/6J mice were equally randomized into Sham group, LPS group, LPS + Alda-1 (an ALDH2 agonist) group, and LPS + Daidzin (an ALDH2 inhibitor) group. After the treatment, the wet/dry lung mass ratio of the mice was measured, and the lung permeability was evaluated with Evans Blue (EB). The lung tissue pathologies were evaluated with HE staining and transmission electron microscopy. Serum levels of 4-hydroxynonenal (4-HNE) were measured with ELISA, and malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined to measure oxidative stress levels. The expressions of ALDH2, ZO-1, Occludin, Mfn2, OPA1, Drp1, Fis1, and nuclear Nrf2 and HO-1 proteins in the lung tissues were detected using Western blotting. The mice with LPS-induced ALI showed severe disruption of the lung tissue structure and endothelial cell tight junctions with significantly increased the lung permeability (P<0.01), increased levels of 4-HNE and MDA (P<0.01), decreased activities of CAT and SOD (P<0.01), lowered expressions of ALDH2, ZO-1, Occludin, Mfn2, and OPA1 proteins, and increased expressions of Drp1, Fis1, and nuclear Nrf2 and HO-1 proteins (P<0.05, P<0.01). Treatment with Alda-1 significantly improved lung tissue pathologies and mitochondrial damage in ALI mice (P<0.01), increased the expressions of ALDH2, ZO-1, Occludin, OPA1, Mfn2, and nuclear Nrf2 and HO-1 proteins, and lowered the expressions of Drp1 and Fis1 proteins (P<0.05, P<0.01). Compared with Alda-1, treatment with Daidzin significantly increased the lung permeability, exacerbated mitochondrial damage, decreased the expression of ALDH2, ZO-1, Occludin, Mfn2, OPA1, and nuclear Nrf2 and HO-1 proteins, and increased expressions of Drp1 and Fis1 proteins (P<0.05, P<0.01). ALDH2 can ameliorate LPSinduced lung endothelial barrier damage in ALI mice by maintaining the balance of mitochondrial dynamics and inhibiting oxidative stress, and the mechanism may be related to the Nrf2/HO-1 pathway.

Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury

BackgroundCerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI.MethodsSH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett’s T3 test (for unequal homogeneity) were used for statistical tests.ResultsERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1S616E or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed.ConclusionInhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model.9RDMMKPHSZ2No4hHf87VtDVideo

The Effect of Resistance Training and Phoenix dactylifera L. Extract on DRP1 and MFN1 Protein Expression, and the Apoptotic Status of Rat Liver: A Safe Alternative to Testosterone Injection

Background: Testosterone enhances athletic performance in men and women, but its consumption has several side effects and is banned from most competitive sports. This study aimed to examine the effect of date palm pollen extracts (DPPE or Phoenix dactylifera L.), testosterone enanthate (T), and resistance training (RT) alone and in combination on hepatic damage and mitochondrial function of adult male rats. Methods: Thirty male Sprague–Dawley rats were randomly divided into six groups: Control (C), RT-treated, DPPE-treated, T-treated, DPPE+RT-treated, and T+RT-treated. The DPPE-treated animals received 100 mg/kg DPPE by gavage (five days/week for four weeks). T was injected subcutaneously into the target groups at a dose of 2 mg/kg daily (five days/week for four weeks). Moreover, the RT program was performed using a vertical ladder with weights (five days/week for four weeks). Results: RT, T, and DPPE significantly reduced collagen degradation, apoptotic cells, dynamin-related protein 1 (DRP1) protein expression, and increased mitofusin-1 (Mfn-1) gene and protein expression in liver tissue. RT with T/DPPE showed a synergic effect regarding present study variables. Conclusions: It seems that DPP, which is a natural compound, has less damaging effects than T on liver tissue. It can be used as a safe alternative to T injection for the enhancement of athletic performance and T deficiency.

Abstract P3206: Sex-specific Differences In Proteasome Activity And Mitochondrial Function In Mouse Hearts Lacking Natriuretic Peptide Receptor A

Objective: Atrial natriuretic peptide (ANP) is a cardiac hormone that plays an important role in cardiovascular function and energy metabolism by signaling through natriuretic peptide receptor A (NPRA). Genetic ablation of NPRA in mice resulted in hypertension, cardiac hypertrophy, and fibrosis. The cardiovascular phenotypes were reported to be more severe in male NPRA-knockout (KO) mice. It is known that reductions in proteasomal activities can lead to mitochondrial dysfunction. Similarly, an imbalance between mitochondrial fission and fusion events can impair mitochondrial function. We hypothesized that alterations in proteasomal function and mitochondrial dynamics might form the basis for phenotype differences reported for NPRA-KO mice. Method/Results: Non-invasive echocardiography, RT-QPCR, Western blotting, and proteasomal assays were performed to evaluate cardiac functions. Preliminary results revealed significant decreases in the activities of two proteasome subunits (β1 and β5) in male and female NPRA-KO hearts compared to sex-matched wild-type (WT) hearts. Notably, the percent-activity reductions were much higher in male NPRA-KO hearts. Western blotting analysis revealed that the expression levels of mitochondrial fusion proteins (Mfn1, Mfn2, and Opa1) were significantly increased in both male and female NPRA-KO hearts compared to WT controls. However, the expression of fission proteins (Drp1 &amp; MFF) was significantly increased only in male NPRA-KO hearts. Consistent with a more imbalance in mitochondrial dynamics in male NPRA-KO hearts, the expression levels of enzymes that promote fatty acid oxidation (ECHS1 and phospho-PDH) were unfavorably altered in male but not in female NPRA-KO hearts compared to controls. In addition, various echocardiographic and electrocardiographic parameters related to the cardiac function such as cardiac output, diastolic volume, left ventricular wall thickness, QRS duration, and stroke volume, were altered significantly more in male than in female NPRA-KO hearts. Conclusion: Our findings suggest that more pronounced sex-specific alterations in proteasome/mitochondrial functions in male NPRA-KO hearts can lead to changes in cardiac metabolism and a severe disease phenotype.

High glucose-induced imbalance of mitochondria-associated ER membranes function promotes RSC96 cell damage.

To investigate the effect of high glucose on mitochondrial-related ER membranes (MAMs) in rat Schwann cells (SCs) and the mechanism of cell injury. SCs (RSC96) cells were used as the control group, and RSC96 cells cultured in a high glucose environment for 48 h were set as the experimental group. The level of intracellular calcium was observed by flow cytometry, and ROS levels were detected by DCFH-DA fluorescent probe. The subcellular structure was observed by transmission electron microscopy, focusing on the morphology of mitochondria and endoplasmic reticulum as well as the formation of MAMs. The expression levels of MAMs-related proteins Mfn2, PERK, VDAC1, and IP3R were detected by Western blot. Compared with the control group, after high glucose-induced cells, the level of calcium ion was significantly increased (p<0.01), the level of ROS was significantly increased (p<0.01), mitochondria and endoplasmic reticulum were damaged, and the number of MAMs was increased (p<0.05). Western blot analysis showed that the expression level of Mfn2 was significantly decreased (p<0.01), and the expression levels of PERK, VDAC1, and IP3R were significantly increased (p<0.01). By inducing the imbalance of MAMs function in SCs, high glucose promotes intracellular calcium overload and leads to cell damage.

A Novel ENU-Induced Mfn2 Mutation Causes Motor Deficits in Mice without Causing Peripheral Neuropathy.

Mitochondrial fission and fusion are required for maintaining functional mitochondria. The mitofusins (MFN1 and MFN2) are known for their roles in mediating mitochondrial fusion. Recently, MFN2 has been implicated in other important cellular functions, such as mitophagy, mitochondrial motility, and coordinating endoplasmic reticulum-mitochondria communication. In humans, over 100 MFN2 mutations are associated with a form of inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). Here we describe an ENU-induced mutant mouse line with a recessive neuromuscular phenotype. Behavioral screening showed progressive weight loss and rapid deterioration of motor function beginning at 8 weeks. Mapping and sequencing revealed a missense mutation in exon 18 of Mfn2 (T1928C; Leu643Pro), within the transmembrane domain. Compared to wild-type and heterozygous littermates, Mfn2L643P/L643P mice exhibited diminished rotarod performance and decreases in activity in the open field test, muscular endurance, mean mitochondrial diameter, sensory tests, mitochondrial DNA content, and MFN2 protein levels. However, tests of peripheral nerve physiology and histology were largely normal. Mutant leg bones had reduced cortical bone thickness and bone area fraction. Together, our data indicate that Mfn2L643P causes a recessive motor phenotype with mild bone and mitochondrial defects in mice. Lack of apparent nerve pathology notwithstanding, this is the first reported mouse model with a mutation in the transmembrane domain of the protein, which may be valuable for researchers studying MFN2 biology.

1599-P: Regulation of Mitochondrial Dynamics Ameliorates Hepatic Steatosis through TFEB Activation

Nonalcoholic fatty liver disease (NAFLD) is a common cause of liver disease that is closely related to metabolic syndromes, obesity, and type 2diabetes. There is strong evidence that mitochondrial dysfunction plays a significant role in the development and progression of NAFLD. Recently, growing evidence suggests that the TFEB plays a pivotal role in mitochondrial dynamics. In this study, we investigated that mitochondrial dynamics play the potential role and mechanisms of TFEB in the amelioration of NAFLD. FFA increased expression of mitochondrial fission proteins p-Drp1, Fis1 and decreased expression of fusion proteins OPA1, Mfn2 and inhibition of TFEB activation. Mitotracker stain significantly increased in the proportion of cells with fragmented mitochondria in FFA-treated cells. HFD-fed mice displayed histology consistent with pathological hepatic steatosis. EM analysis indicated that HFD-fed mitochondria tended to be shorter and more round than ND-fed mitochondria. Western blot analyses reveal increased expression of the fission proteins, p-Drp1, Fis1, and significantly decreased in the fusion protein, Mfn2 and OPA1 in HFD-fed group. In vivo Tfeb knockout dramatically increased hepatic fat accumulation, liver ballooning in HFD-fed mice. EM analysis showed that HFD-fed Tfeb knockout mice had more rounded and swollen mitochondria, indicating that TFEB was dependent on the regulation of mitochondrial fragmentation. In vivo Tfeb knockout reveal significantly increased expression of the fission proteins in HFD-fed group. To demonstrated the direct regulatory mechanism of mitochondrial dynamics and TFEB activation, we used siRNA DRP1 and Mdivi-1. Inhibition of Drp1 promotes mitochondrial fusion, increased expression of fusion protein and reduced fat accumulation and activated TFEB. These results suggest a role in improving hepatic steatosis through regulation of TFEB-mediated mitochondrial dynamics in NAFLD. Disclosure J.Yoo: Other Relationship; American Diabetes Association. Y.Hwang: None. Funding National Research Foundation of Korea (2022R1I1A1A01069899)

Attenuation of amyloid-β-induced mitochondrial dysfunction by active components of anthocyanins in HT22 neuronal cells.

Alzheimer's disease (AD) is a common form of neurodegenerative disease in the elderly. Amyloid-β (Aβ)-associated neurotoxicity is an important component of the neurodegenerative change in AD. Recent studies have revealed a beneficial effect of anthocyanins in improving learning and memory in AD animal models. Using cultured HT22 mouse hippocampal neuronal cells as an in vitro model, we examined in this study the protective effect of ten pure components of anthocyanins against Aβ 42-induced cytotoxicity and also investigated the mechanism of their protective effects. We found that treatment of HT22 cells with the pure components of anthocyanins dose-dependently rescued Aβ 42-induced cytotoxicity, with slightly different potencies. Using petunidin as a representative compound, we found that it enhanced mitochondrial homeostasis and function in Aβ 42-treated HT22 cells. Mechanistically, petunidin facilitated β-catenin nuclear translocation and enhanced the interaction between β-catenin and TCF7, which subsequently upregulated mitochondrial homeostasis-related protein Mfn2, thereby promoting restoration of mitochondrial homeostasis and function in Aβ 42-treated HT22 cells. Together, these results reveal thatthe pure components of anthocyanins have a strong protective effect in HT22 cells against Aβ 42-induced cytotoxicity by ameliorating mitochondrial homeostasis and function in a β-catenin/TCF-dependent manner.

EXPRESSION OF MITOCHONDRIAL DYNAMIC MARKERS IN ADENOID CYSTIC CARCINOMA

Objective This study aimed to investigate the prognostic significance of mitochondrial dynamic markers in adenoid cystic carcinoma (AdCC). Study Design Fifty-seven formalin-fixed paraffin-embedded cases were retrieved and disposed in a tissue microarray. Histological sections were submitted to immunohistochemical reactions against AMT, DRP1, FIS1, MFN1, MFN2, and OPA1 proteins. Clinical data were retrieved from the patients’ medical files, including specific and disease-free survival data. Results It was observed that 50.9% of the cases were strongly positive for AMT and DRP1, while 49.1%, 21.1%, 22.8%, and 24.6% were strongly positive for FIS1, MFN1, MFN2, and OPA1, respectively. Reactions were observed in both epithelial and myoepithelial components of the tumor. The higher expression of MFN2 was associated with a solid microscopic pattern (p = .016). DRP1 overexpression showed a trend towards a shorter overall survival (p = .054), while negative/weak OPA1 showed a trend towards a lower DFS (p=.051) in the univariate analysis, but no mitochondrial marker represented an independent prognostic determinant under multivariate analysis. Conclusion Mitochondrial dynamics markers do not seem to carry a prognostic significance for AdCC patients, but these proteins may play an important role in its pathogenesis. Support: FAPEMIG, CAPES, CNPq. This study aimed to investigate the prognostic significance of mitochondrial dynamic markers in adenoid cystic carcinoma (AdCC). Fifty-seven formalin-fixed paraffin-embedded cases were retrieved and disposed in a tissue microarray. Histological sections were submitted to immunohistochemical reactions against AMT, DRP1, FIS1, MFN1, MFN2, and OPA1 proteins. Clinical data were retrieved from the patients’ medical files, including specific and disease-free survival data. It was observed that 50.9% of the cases were strongly positive for AMT and DRP1, while 49.1%, 21.1%, 22.8%, and 24.6% were strongly positive for FIS1, MFN1, MFN2, and OPA1, respectively. Reactions were observed in both epithelial and myoepithelial components of the tumor. The higher expression of MFN2 was associated with a solid microscopic pattern (p = .016). DRP1 overexpression showed a trend towards a shorter overall survival (p = .054), while negative/weak OPA1 showed a trend towards a lower DFS (p=.051) in the univariate analysis, but no mitochondrial marker represented an independent prognostic determinant under multivariate analysis. Mitochondrial dynamics markers do not seem to carry a prognostic significance for AdCC patients, but these proteins may play an important role in its pathogenesis. Support: FAPEMIG, CAPES, CNPq.

Mdivi-1 Rescues Memory Decline in Scopolamine-Induced Amnesic Male Mice by Ameliorating Mitochondrial Dynamics and Hippocampal Plasticity.

Memory loss, often known as amnesia, is common in the elderly population and refers to forgetting facts and experiences. It is associated with increased mitochondrial fragmentation, though the contribution of mitochondrial dynamics in amnesia is poorly understood. Therefore, the present study is aimed at elucidating the role of Mdivi-1 in mitochondrial dynamics, hippocampal plasticity, and memory during scopolamine(SC)-induced amnesia. The findings imply that Mdivi-1 significantly increased the expression of Arc and BDNF proteins in the hippocampus of SC-induced amnesic mice, validating improved recognition and spatial memory. Moreover, an improved mitochondrial ultrastructure was attributed to a decline in the percentage of fragmented and spherical-shaped mitochondria after Mdivi-1 treatment in SC-induced mice. The significant downregulation of p-Drp1 (S616) protein and upregulation of Mfn2, LC3BI, and LC3BII proteins in Mdivi-1-treated SC-induced mice indicated a decline in fragmented mitochondrial number and healthy mitochondrial dynamics. Mdivi-1 treatment alleviated ROS production and Caspase-3 activity and elevated mitochondrial membrane potential, Vdac1 expression, ATP production, and myelination, resulting in reduced neurodegeneration in SC mice. Furthermore, the decline of pro-apoptotic protein cytochrome-c and increase of anti-apoptotic proteins Procaspase-9 and Bcl-2 in Mdivi-1-treated SC-induced mice suggested improved neuronal health. Mdivi-1 also increased the dendritic arborization and spine density, which was further corroborated by increased expression of synaptophysin and PSD95. In conclusion, the current study suggests that Mdivi-1 treatment improves mitochondrial ultrastructure and function through the regulation of mitochondrial dynamics. These changes further improve neuronal cell density, myelination, dendritic arborization, and spine density, decrease neurodegeneration, and improve recognition and spatial memory. Schematic presentation depicts that Mdivi-1 rescues memory decline in scopolamine-induced amnesic male mice by ameliorating mitochondrial dynamics and hippocampal plasticity.

Metformin protects against retinal ischemia/reperfusion injury through AMPK-mediated mitochondrial fusion

Retinal ischemia/reperfusion (I/R) injury is a common pathological process responsible for cellular damage in glaucoma, diabetic retinopathy and hypertensive retinopathy. Metformin is a biguanide drug that exerts strong effects on multiple diseases. This study aims to evaluate the protective effect of metformin against retinal I/R injury and its underlying mechanism. I/R induced reduction in retina thickness and cell number in ganglion cell layer, and metformin alleviated I/R-induced retinal injury. Both retinal I/R and simulated ischemia/reperfusion (SIR) in R28 cells down-regulated expression of mitochondrial fusion protein Mfn2 and OPA1, which led to mitochondrial fission. Metformin also alleviated damage in R28 cells, and reversed the alteration in Mfn2 and OPA1, mitochondrial fission and mitochondrial membrane potential (MMP) disruption-induced by I/R or SIR as well. Intriguingly, inhibition of AMPK by compound C or siRNA prevented metformin-mediated up-regulation of Mfn2 and OPA1. Compound C and knockdown of Mfn2 or OPA1 dramatically alleviated the protective effect of metformin against intracellular ROS generation, MMP disruption, mitochondrial fission and loss of RGCs in ganglion cell layer induced by SIR or I/R. Moreover, scavenging mitochondrial ROS (mito-ROS) by mito-TEMPO exerted the similar protection against I/R-induced retinal injury or SIR-induced damage in R28 cells as metformin. Our data show for the first time that metformin protects against retinal I/R injury through AMPK-mediated mitochondrial fusion and the decreased mito-ROS generation. These findings might also repurpose metformin as a therapeutic agent for retinal I/R injury.