Fueling the heartbeat: Dynamic regulation of intracellular ATP during excitation-contraction coupling in ventricular myocytes.

The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca2+ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]i. Notably, diastolic [ATP]i was <1 mM, which is eightfold to 10-fold lower than previously estimated. Accordingly, ATP-sensitive K+ (KATP) channels were active at physiological [ATP]i. Cells exhibited two distinct types of ATP fluctuations during an action potential: net increases (Mode 1) or decreases (Mode 2) in [ATP]i. Mode 1 [ATP]i increases necessitated Ca2+ entry and release from the sarcoplasmic reticulum (SR) and were associated with increases in mitochondrial Ca2+. By contrast, decreases in mitochondrial Ca2+ accompanied Mode 2 [ATP]i decreases. Down-regulation of the protein mitofusin 2 reduced the magnitude of [ATP]i fluctuations, indicating that SR-mitochondrial coupling plays a crucial role in the dynamic control of ATP levels. Activation of ß-adrenergic receptors decreased [ATP]i, underscoring the energetic impact of this signaling pathway. Finally, our work suggests that cross-bridge cycling is the largest consumer of ATP in a ventricular myocyte during an action potential. These findings provide insights into the energetic demands of EC coupling and highlight the dynamic nature of ATP concentrations in cardiac muscle.

Mfn2-dependent fusion pathway of PE-enriched micron-sized vesicles.

Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.

Mitofusin 2 Sustains the Axonal Mitochondrial Network to Support Presynaptic Ca2+ Homeostasis and the Synaptic Vesicle Cycle in Rat Hippocampal Axons.

Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metabolite biosynthesis. The trafficking and function of these organelles are particularly important in neurons, with impaired mitochondrial function or altered morphology observed in every neurodegenerative disorder studied. While mitochondrial biosynthetic products play a crucial role in maintaining cellular function, their resulting byproducts can have negative consequences. Thus, organelle quality control (QC) mechanisms that maintain mitochondrial function are imperative to restrict destructive signaling cascades in the cell. Axons are particularly sensitive to damage, and there is little consensus regarding the mechanisms that mediate mitochondrial QC in this compartment. Here, we first investigated the unstressed behavior of mitochondria in rat hippocampal neurons of mixed sex, focusing on mitochondrial trafficking and fusion to better understand potential QC mechanisms. We observed size and redox asymmetry of mitochondrial traffic in axons, suggesting an active QC mechanism in this compartment. We also document biochemical complementation upon the fusion and fission of axonal mitochondria. Eliminating fusion by knocking down the neuronal mitochondrial fusion protein mitofusin 2 (MFN2) reduced the rates of axonal mitochondrial trafficking and fusion, decreased the levels of synaptic vesicle (SV) proteins, inhibited exocytosis, and impaired SV recruitment from the reserve pool during extended stimulation. MFN2 knockdown also resulted in presynaptic Ca2+ dyshomeostasis. Remarkably, upon MFN2 knockdown, presynaptic mitochondria sequestered Ca2+ more efficiently, effectively limiting presynaptic Ca2+ transients during stimulation. These results support an active mitochondrial trafficking and fusion-related QC process that supports presynaptic Ca2+ handling and the SV cycle.SIGNIFICANCE STATEMENT Decreased or altered mitochondrial function is observed in many disease states. All neurodegenerative diseases co-present with some sort of mitochondrial abnormality. Therefore, identifying quality control mechanisms that sustain the mitochondrial network in neurons, and particularly in axons, is of significant interest. The response of axonal mitochondria to acutely applied toxins or injury has been studied in detail. Although informative, the response of neurons to these insults might not be physiologically relevant, so it is crucial to also study the basal behavior of axonal mitochondria. Here, we use fluorescent biosensors to investigate the mitochondrial network in neurons and examine the role of mitofusin 2 in maintaining the axonal mitochondrial network and in supporting the synaptic vesicle cycle.

Charcot-Marie-Tooth type 2A in vivo models: Current updates.

Charcot-Marie-Tooth type 2A (CMT2A) is an inherited sensorimotor neuropathy associated with mutations within the Mitofusin 2 (MFN2) gene. These mutations impair normal mitochondrial functioning via different mechanisms, disturbing the equilibrium between mitochondrial fusion and fission, of mitophagy and mitochondrial axonal transport. Although CMT2A disease causes a significant disability, no resolutive treatment for CMT2A patients to date. In this context, reliable experimental models are essential to precisely dissect the molecular mechanisms of disease and to devise effective therapeutic strategies. The most commonly used models are either in vitro or in vivo, and among the latter murine models are by far the most versatile and popular. Here, we critically revised the most relevant literature focused on the experimental models, providing an update on the mammalian models of CMT2A developed to date. We highlighted the different phenotypic, histopathological and molecular characteristics, and their use in translational studies for bringing potential therapies from the bench to the bedside. In addition, we discussed limitations of these models and perspectives for future improvement.

Charcot-Marie-tooth disease type 2A: An update on pathogenesis and therapeutic perspectives.

Mutations in the gene encoding MFN2 have been identified as associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a neurological disorder characterized by a broad clinical phenotype involving the entire nervous system. MFN2, a dynamin-like GTPase protein located on the outer mitochondrial membrane, is well-known for its involvement in mitochondrial fusion. Numerous studies have demonstrated its participation in a network crucial for various other mitochondrial functions, including mitophagy, axonal transport, and its controversial role in endoplasmic reticulum (ER)-mitochondria contacts. Considerable progress has been made in the last three decades in elucidating the disease pathogenesis, aided by the generation of animal and cellular models that have been instrumental in studying disease physiology. A review of the literature reveals that, up to now, no definitive pharmacological treatment for any CMT2A variant has been established; nonetheless, recent years have witnessed substantial progress. Many treatment approaches, especially concerning molecular therapy, such as histone deacetylase inhibitors, peptide therapy to increase mitochondrial fusion, the new therapeutic strategies based on MF1/MF2 balance, and SARM1 inhibitors, are currently in preclinical testing. The literature on gene silencing and gene replacement therapies is still limited, except for a recent study by Rizzo et al.(Rizzo et al., 2023), which recently first achieved encouraging results in in vitro and in vivo models of the disease. The near-future goal for these promising therapies is to progress to the stage of clinical translation.

Metformin normalizes mitochondrial function to delay astrocyte senescence in a mouse model of Parkinson's disease through Mfn2-cGAS signaling.

BACKGROUND: Senescent astrocytes play crucial roles in age-associated neurodegenerative diseases, including Parkinson's disease (PD). Metformin, a drug widely used for treating diabetes, exerts longevity effects and neuroprotective activities. However, its effect on astrocyte senescence in PD remains to be defined. METHODS: Long culture-induced replicative senescence model and 1-methyl-4-phenylpyridinium/α-synuclein aggregate-induced premature senescence model, and a mouse model of PD were used to investigate the effect of metformin on astrocyte senescence in vivo and in vitro. Immunofluorescence staining and flow cytometric analyses were performed to evaluate the mitochondrial function. We stereotactically injected AAV carrying GFAP-promoter-cGAS-shRNA to mouse substantia nigra pars compacta regions to specifically reduce astrocytic cGAS expression to clarify the potential molecular mechanism by which metformin inhibited the astrocyte senescence in PD. RESULTS: We showed that metformin inhibited the astrocyte senescence in vitro and in PD mice. Mechanistically, metformin normalized mitochondrial function to reduce mitochondrial DNA release through mitofusin 2 (Mfn2), leading to inactivation of cGAS-STING, which delayed astrocyte senescence and prevented neurodegeneration. Mfn2 overexpression in astrocytes reversed the inhibitory role of metformin in cGAS-STING activation and astrocyte senescence. More importantly, metformin ameliorated dopamine neuron injury and behavioral deficits in mice by reducing the accumulation of senescent astrocytes via inhibition of astrocytic cGAS activation. Deletion of astrocytic cGAS abolished the suppressive effects of metformin on astrocyte senescence and neurodegeneration. CONCLUSIONS: This work reveals that metformin delays astrocyte senescence via inhibiting astrocytic Mfn2-cGAS activation and suggest that metformin is a promising therapeutic agent for age-associated neurodegenerative diseases.

Matrix metalloproteinase-2 proteolyzes mitofusin-2 and impairs mitochondrial function during myocardial ischemia-reperfusion injury.

During myocardial ischemia and reperfusion (IR) injury matrix metalloproteinase-2 (MMP-2) is rapidly activated in response to oxidative stress. MMP-2 is a multifunctional protease that cleaves both extracellular and intracellular proteins. Oxidative stress also impairs mitochondrial function which is regulated by different proteins, including mitofusin-2 (Mfn-2), which is lost in IR injury. Oxidative stress and mitochondrial dysfunction trigger the NLRP3 inflammasome and the innate immune response which invokes the de novo expression of an N-terminal truncated isoform of MMP-2 (NTT-MMP-2) at or near mitochondria. We hypothesized that MMP-2 proteolyzes Mfn-2 during myocardial IR injury, impairing mitochondrial function and enhancing the inflammasome response. Isolated hearts from mice subjected to IR injury (30 min ischemia/40 min reperfusion) showed a significant reduction in left ventricular developed pressure (LVDP) compared to aerobically perfused hearts. IR injury increased MMP-2 activity as observed by gelatin zymography and increased degradation of troponin I, an intracellular MMP-2 target. MMP-2 preferring inhibitors, ARP-100 or ONO-4817, improved post-ischemic recovery of LVDP compared to vehicle perfused IR hearts. In muscle fibers isolated from IR hearts the rates of mitochondrial oxygen consumption and ATP production were impaired compared to those from aerobic hearts, whereas ARP-100 or ONO-4817 attenuated these reductions. IR hearts showed higher levels of NLRP3, cleaved caspase-1 and interleukin-1ß in the cytosolic fraction, while the mitochondria-enriched fraction showed reduced levels of Mfn-2, compared to aerobic hearts. ARP-100 or ONO-4817 attenuated these changes. Co-immunoprecipitation showed that MMP-2 is associated with Mfn-2 in aerobic and IR hearts. ARP-100 or ONO-4817 also reduced infarct size and cell death in hearts subjected to 45 min ischemia/120 min reperfusion. Following myocardial IR injury, impaired contractile function and mitochondrial respiration and elevated inflammasome response could be attributed, at least in part, to MMP-2 activation, which targets and cleaves mitochondrial Mfn-2. Inhibition of MMP-2 activity protects against cardiac contractile dysfunction in IR injury in part by preserving Mfn-2 and suppressing inflammation.

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

BACKGROUND: Maladaptive 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 RESULTS: We 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. CONCLUSIONS: The 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.

DNA Methylation-Mediated Mfn2 Gene Regulation in the Brain: A Role in Brain Trauma-Induced Mitochondrial Dysfunction and Memory Deficits.

Repeated mild traumatic brain injuries (rMTBI) affect mitochondrial homeostasis in the brain. However, mechanisms of long-lasting neurobehavioral effects of rMTBI are largely unknown. Mitofusin 2 (Mfn2) is a critical component of tethering complexes in mitochondria-associated membranes (MAMs) and thereby plays a pivotal role in mitochondrial functions. Herein, we investigated the implications of DNA methylation in the Mfn2 gene regulation, and its consequences on mitochondrial dysfunction in the hippocampus after rMTBI. rMTBI dramatically reduced the mitochondrial mass, which was concomitant with decrease in Mfn2 mRNA and protein levels. DNA hypermethylation at the Mfn2 gene promoter was observed post 30 days of rMTBI. The treatment of 5-Azacytidine, a pan DNA methyltransferase inhibitor, normalized DNA methylation levels at Mfn2 promoter, which further resulted into restoration of Mfn2 function. The normalization of Mfn2 function was well correlated with recovery in memory deficits in rMTBI-exposed rats. Since, glutamate excitotoxicity serves as a primary insult after TBI, we employed in vitro model of glutamate excitotoxicity in human neuronal cell line SH-SY5Y to investigate the causal epigenetic mechanisms of Mfn2 gene regulation. The glutamate excitotoxicity reduced Mfn2 levels via DNA hypermethylation at Mfn2 promoter. Loss of Mfn2 caused significant surge in cellular and mitochondrial ROS levels with lowered mitochondrial membrane potential in cultured SH-SY5Y cells. Like rMTBI, these consequences of glutamate excitotoxicity were also prevented by 5-AzaC pre-treatment. Therefore, DNA methylation serves as a vital epigenetic mechanism involved in Mfn2 expression in the brain; and this Mfn2 gene regulation may play a pivotal role in rMTBI-induced persistent cognitive deficits. Closed head weight drop injury method was employed to induce repeated mild traumatic brain (rMTBI) in jury in adult, male Wistar rats. rMTBI causes hyper DNA methylation at the Mfn2 promoter and lowers the Mfn2 expression triggering mitochondrial dysfunction. However, the treatment of 5-azacytidine normalizes DNA methylation at the Mfn2 promoter and restores mitochondrial function.

Mitofusin 1 overexpression rescues the abnormal mitochondrial dynamics caused by the Mitofusin 2 K357T mutation in vitro.

BACKGROUND AND AIMS: Mitofusin 1 (MFN1) and MFN2 are outer mitochondrial membrane fusogenic proteins regulating mitochondrial network morphology. MFN2 mutations cause Charcot-Marie-Tooth type 2A (CMT2A), an axonal neuropathy characterized by mitochondrial fusion defects, which in the case of a GTPase domain mutant, were rescued following wild-type MFN1/2 (MFN1/2WT ) overexpression. In this study, we compared the therapeutic efficiency between MFN1WT and MFN2WT overexpression in correcting mitochondrial defects induced by the novel MFN2K357T mutation located in the highly conserved R3 region. METHODS: Constructs expressing either MFN2K357T , MFN2WT , or MFN1WT under the ubiquitous chicken ß-actin hybrid (CBh) promoter were generated. Flag or myc tag was used for their detection. Differentiated SH-SY5Y cells were single transfected with MFN1WT , MFN2WT , or MFN2K357T , as well as double transfected with MFN2K357T /MFN2WT or MFN2K357T /MFN1WT . RESULTS: SH-SY5Y cells transfected with MFN2K357T exhibited severe perinuclear mitochondrial clustering with axon-like processes devoid of mitochondria. Single transfection with MFN1WT resulted in a more interconnected mitochondrial network than transfection with MFN2WT , accompanied by mitochondrial clusters. Double transfection of MFN2K357T with either MFN1WT or MFN2WT resolved the mutant-induced mitochondrial clusters and led to detectable mitochondria throughout the axon-like processes. MFN1WT showed higher efficacy than MFN2WT in rescuing these defects. INTERPRETATION: These results further demonstrate the higher potential of MFN1WT over MFN2WT overexpression to rescue CMT2A-induced mitochondrial network abnormalities due to mutations outside the GTPase domain. This higher phenotypic rescue conferred by MFN1WT , possibly due to its higher mitochondrial fusogenic ability, may be applied to different CMT2A cases regardless of the MFN2 mutation type.

High-fat diet causes mitochondrial damage and downregulation of mitofusin-2 and optic atrophy-1 in multiple organs.

High-fat consumption promotes the development of obesity, which is associated with various chronic illnesses. Mitochondria are the energy factories of eukaryotic cells, maintaining self-stability through a fine-tuned quality-control network. In the present study, we evaluated high-fat diet (HFD)-induced changes in mitochondrial ultrastructure and dynamics protein expression in multiple organs. C57BL/6J male mice were fed HFD or normal diet (ND) for 24 weeks. Compared with ND-fed mice, HFD-fed mice exhibited increased body weight, cardiomyocyte enlargement, pulmonary fibrosis, hepatic steatosis, renal and splenic structural abnormalities. The cellular apoptosis of the heart, liver, and kidney increased. Cellular lipid droplet deposition and mitochondrial deformations were observed. The proteins related to mitochondrial biogenesis (TFAM), fission (DRP1), autophagy (LC3 and LC3-II: LC3-I ratio), and mitophagy (PINK1) presented different changes in different organs. The mitochondrial fusion regulators mitofusin-2 (MFN2) and optic atrophy-1 (OPA1) were consistently downregulated in multiple organs, even the spleen. TOMM20 and ATP5A protein were enhanced in the heart, skeletal muscle, and spleen, and attenuated in the kidney. These results indicated that high-fat feeding caused pathological changes in multiple organs, accompanied by mitochondrial ultrastructural damage, and MFN2 and OPA1 downregulation. The mitochondrial fusion proteins may become promising targets and/or markers for treating metabolic disease.

Mitofusin-2 in cancer: Friend or foe?

Cancer is a category of disorders characterized by excessive cell proliferation with the ability to infiltrate or disseminate to other organs of the body. Mitochondrial dysfunction, as one of the most prominent hallmarks of cancer cells, has been related to the onset and development of various cancers. Mitofusin 2 (MFN2) is a major mediator of mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria interaction, mitophagy and axonal transport of mitochondria. Available data have shown that MFN2, which its alterations have been associated with mitochondrial dysfunction, could affect cancer initiation and progression. In fact, it showed that MFN2 may have a double-edged sword effect on cancer fate. Precisely, it demonstrated that MFN2, as a tumor suppressor, induces cancer cell apoptosis and inhibits cell proliferation via Ca2+ and Bax-mediated apoptosis and increases P21 and p27 levels, respectively. It also could suppress cell survival via inhibiting PI3K/Akt, Ras-ERK1/2-cyclin D1 and mTORC2/Akt signaling pathways. On the other hand, MFN2, as an oncogene, could increase cancer invasion via snail-mediated epithelial-mesenchymal transition (EMT) and in vivo tumorigenesis. While remarkable progress has been achieved in recent decades, further exploration is required to elucidate whether MFN2 could be a friend or it's an enemy. This study aimed to highlight the different functions of MFN2 in various cancers.

Neural cell-derived plasma exosome protein abnormalities implicate mitochondrial impairment in first episodes of psychosis.

Neuroprotective and other functional proteins of mitochondria were quantified in extracts of plasma neural-derived exosomes from ten first-episode psychosis (FP) patients and ten matched psychiatrically normal controls (ctls). Astrocyte-derived extracellular vesicles (ADEVs) and neuron-derived extracellular vesicles (NDEVs) were immunoabsorbed separately from physically precipitated plasma total EVs. Extracted mitochondrial ATP synthase was specifically immunofixed to plastic wells for quantification of catalytic activity based on conversion of NADH to NAD+ . Other extracted mitochondrial functional proteins were quantified by ELISAs. All protein levels were normalized with EV content of the CD81 exosome marker. FP patient ADEV level but not NDEV level of mitochondrial ATP synthase activity was significantly lower than that of ctls. FP patient ADEV and NDEV levels of the functionally critical mitochondrial proteins mitofusin 2 and cyclophilin D, but not of transcription factor A of mitochondria, and of the mitochondrial short open-reading frame neuroprotective and metabolic regulatory peptides humanin and MOTS-c were significantly lower than those of ctls. In contrast, FP patient NDEV, but not ADEV, level of the mitochondrial-tethering protein syntaphilin, but not of myosin VI, was significantly higher than that of ctls. The distinctively different neural levels of some mitochondrial proteins in FP patients than ctls now should be correlated with diverse clinical characteristics. Drugs that increase depressed levels of proteins and mimetics of deficient short open-reading frame peptides may be of therapeutic value in early phases of schizophrenia.

Mitofusin 2 confers the suppression of microglial activation by cannabidiol: Insights from in vitro and in vivo models.

Currently, there is increasing attention on the regulatory effects of cannabidiol (CBD) on the inflammatory response and the immune system. However, the mechanisms have not yet been completely revealed. Mitofusin 2 (Mfn2) is a mitochondrial fusion protein involved in the inflammatory response. Here, we investigated whether Mfn2 confers the anti-inflammatory effects of CBD. We found that treatment with CBD decreased the levels of tumor necrosis factor α, interleukin 6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and ionized calcium-binding adaptor molecule-1 (Iba1) in lipopolysaccharide (LPS)-challenged microglia. CBD also significantly suppressed the increase in reactive oxygen species (ROS) and the decline of mitochondrial membrane potential in BV-2 cells subjected to LPS. Interestingly, CBD treatment increased the expression of Mfn2, while knockdown of Mfn2 blocked the effect of CBD. By contrast, overexpression of Mfn2 reversed the increase in the levels of iNOS, COX-2, and Iba1 induced by Mfn2 small interfering RNA. In mice challenged with LPS, we found that CBD ameliorated the anxiety responses and cognitive deficits, increased the level of Mfn2, and decreased the expression of Iba1. Since neuro-inflammation and microglial activation are the common events that are observed in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, we treated EAE mice with CBD. Mice that received CBD showed amelioration of clinical signs, reduced inflammatory response, and increased myelin basic protein level. Most importantly, the adeno-associated virus delivery of short hairpin RNA against Mfn2 reversed the protective effects of CBD. Altogether, these results indicate that Mfn2 is an essential immunomodulator conferring the anti-inflammatory effects of CBD. Our results also shed new light on the mechanisms underlying the protective effects of CBD against inflammatory diseases including multiple sclerosis.

Activation of endoplasmic reticulum-mitochondria coupling drives copper-induced autophagy in duck renal tubular epithelial cells.

Copper (Cu) as a transition metal can be toxic to public and ecosystem health at high level, but the specific mechanism of Cu-evoked nephrotoxicity remains elusive. Here, we first revealed the crosstalk between mitofusin2 (Mfn2)-dependent mitochondria-associated endoplasmic reticulum membrane (MAM) dynamics and autophagy in duck renal tubular epithelial cells under Cu exposure. Primary duck renal tubular epithelial cells were treated with 100 and 200 µM Cu sulfate for 12 h and exposed to lentivirus to deliver mitofusin2 (Mfn2). We found that excessive Cu disrupted MAM integrity, decreased the mitochondrial calcium level, co-localization of IP3R and VDAC1, the mRNA levels of PACS2, Mfn2, IP3R and MCU, and Mfn2 and VDAC1 protein levels, causing MAM dysfunction. Furthermore, Mfn2 overexpression ameliorated Cu-induced MAM dysfunction, and increased Cu-evoked autophagy in duck renal tubular epithelial cells accompanied with the elevation of autophagosomes number, ROS level, LC3 puncta, Atg5 and LC3B mRNA levels, and Beclin1, Atg14, LC3BII/LC3BI protein levels. Accordingly, our data proved that excessive Cu could trigger MAM dysfunction and autophagy in duck renal tubular epithelial cells, and Cu-induced autophagy could be activated through Mfn2-dependent MAM, providing evidence on the toxicological exploration mechanisms of Cu.

Arsenic induces ferroptosis and acute lung injury through mtROS-mediated mitochondria-associated endoplasmic reticulum membrane dysfunction.

The goal of this study was to analyze whether mitochondria-associated endoplasmic reticulum membrane (MAMs) dysfunction mediated arsenic (As)-evoked pulmonary ferroptosis and acute lung injury (ALI). As exposure led to alveolar structure damage, inflammatory cell infiltration and pulmonary function decline in mice. Ferritin, the marker of iron overload, was increased, GPX4, the index of lipid peroxidation, was decreased in As-exposed lungs and pulmonary epithelial cells (MLE-12). Pretreatment with ferrostatin-1 (Fer-1), the inhibitor of ferroptosis, alleviated As-evoked ALI. In addition, As-induced non-heme iron deposition was inhibited in Fer-1 pretreated-mice. Moreover, As-triggered mitochondria damage and ferroptosis were mitigated in Fer-1 pretreated-MLE-12 cells. Mechanistically, PERK phosphorylation and mitofusin-2 (Mfn-2) reduction was observed in As-exposed MLE-12 cells and mice lungs. Additionally, the interaction between PERK and Mfn-2 was downregulated and MAMs dysfunction was observed in As-exposed MLE-12 cells. Intriguingly, PERK inhibitor and Mfn-2-overexpression all mitigated As-induced ferroptosis in MLE-12 cells. Additionally, CLPP and mtHSP70, the markers of mitochondrial stress, were upregulated, mitochondrial ROS (mtROS) was elevated, mitochondrial membrane potential (MMP) and ATP were decreased in As-exposed MLE-12 cells. Mitoquinone mesylate (MitoQ), a novel mitochondrial-targeted antioxidant, alleviated As-induced excess mtROS, mitochondrial stress, MAMs dysfunction in pulmonary epithelial cells. Similarly, in vivo experiments indicated that MitoQ pretreatment countered As-induced pulmonary ferroptosis and ALI. These data indicated that mtROS-initiated MAMs dysfunction is, at least partially, implicated in As-evoked ferroptosis and ALI.

[Tianma Gouteng Decoction regulates MFN2 expression to delay vascular aging in spontaneously hypertensive rats].

We studied the effect of Tianma Gouteng Decoction on vascular aging in spontaneously hypertensive rats(SHRs) to explore the molecular mechanism of the decoction in improving arterial vascular aging by regulating the expression of mitofusin 2(MFN2).Twenty 64-weeks-old SHRs were randomly assigned into the aging group and the treatment group(Tianma Gouteng Decoction 5.48 mg·kg~(-1)).Wistar-Kyoto(WKY) rats of 64 weeks old were taken as the normal group and SHR rats of 14 weeks old as the young group.The intervention with Tianma Gouteng Decoction lasted for 12 weeks.We then employed HE staining and Masson staining to observe the morphology of thoracic aorta under an electron microscope and measured the malondialdehyde(MDA) content, superoxide dismutase(SOD) activity, respiratory chain complex Ⅲ level, and thioredoxin peroxidase(TPX) activity.The vascular aging was detected via the biomarker senescence-associated beta-galactosidase(SA-ß-Gal).The expression levels of MFN2 and aging marker proteins silent information regulator 1(SIRT1), Klotho, p21, and p53 in thoracic aorta were detected by immunohistochemistry/fluorescence, qRT-PCR, and Western blot.Compared with the young group and the normal group, the aging group had risen blood pressure, lumen stenosis caused by thickened intima of blood vessels, decreased SOD and TPX activities, increased MDA and mitochondrial respiratory chain complex Ⅲ levels, down-regulated expression of MFN2, SIRT1, and Klotho, and up-regulated expression of p21 and p53(P<0.01 or P<0.05).The treatment with Tianma Gouteng Decoction significantly lowered blood pressure, mitigated vascular intimal thickening, increased SOD and TPX activities, and decreased MDA and mitochondrial respiratory chain complex Ⅲ levels, thus alleviating vascular aging.At the same time, the decoction up-regulated the expression of MFN2, SIRT1, and Klotho, while down-regulated that of p21 and p53(P<0.01 or P<0.05).In summary, Tianma Gouteng Decoction can significantly delay the vascular aging in hypertension.Specifically, it may up-regulate the expression of MFN2 and regulate the expression of aging-related proteins to alleviate oxidative stress.

Mycobacterium tuberculosis infection up-regulates MFN2 expression to promote NLRP3 inflammasome formation.

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1ß secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.

Mfn2 is critical for brown adipose tissue thermogenic function.

Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine-tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin-resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose-specific Mfn2 knockout (Mfn2-adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2-adKO mice were protected from high-fat diet-induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole-body energy homeostasis.

Effect of photobiomodulation on mitochondrial dynamics in peripheral nervous system in streptozotocin-induced type 1 diabetes in rats.

There is no effective treatment to halt peripheral nervous system damage in diabetic peripheral neuropathy. Mitochondria have been at the center of discussions as important factors in the development of neuropathy in diabetes. Photobiomodulation has been gaining clinical acceptance as it shows beneficial effects on a variety of nervous system disorders. In this study, the effects of photobiomodulation (904 nm, 45 mW, 6.23 J/cm2, 0.13 cm2, 60 ns pulsed time) on mitochondrial dynamics were evaluated in an adult male rat experimental model of streptozotocin-induced type 1 diabetes. Results presented here indicate that photobiomodulation could have an important role in preventing or reversing mitochondrial dynamics dysfunction in the course of peripheral nervous system damage in diabetic peripheral neuropathy. Photobiomodulation showed its effects on modulating the protein expression of mitofusin 2 and dynamin-related protein 1 in the sciatic nerve and in the dorsal root ganglia neurons of streptozotocin-induced type 1 diabetes in rats.