MFN2 retrotranslocation boosts mitophagy by uncoupling mitochondria from the ER

Mitochondrial damage triggers mitochondrial quality control pathways, which act to ensure the health of the mitochondrial network. The turnover of damaged mitochondria by mitophagy is initiated by the Parkinson disease-linked genes PRKN and PINK1, and we recently investigated the role that interorganellar contact sites between the endoplasmic reticulum (ER) and the outer mitochondrial membrane (OMM) play in this pathway. In this punctum, we summarize our findings that show that the ER-OMM tether MFN2 acts as a suppressor of mitophagy through its ability to link the OMM to the ER, potentially limiting the accessibility of other ubiquitination substrates to PINK1 and PRKN. PINK1, PRKN and the AAA-ATPase VCP disrupt contact between mitochondria and the ER via MFN2 ubiquitination, retrotranslocation and turnover from the mitochondrial membrane. Our study provides insight into the role of OMM remodeling in mitophagy.     

MFN2 Couples Glutamate Excitotoxicity and Mitochondrial Dysfunction in Motor Neurons

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca²⁺-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.     

The soluble form of Bax regulates mitochondrial fusion via MFN2 homotypic complexes

In mammals, fusion of the mitochondrial outer membrane is controlled by two DRPs, MFN1 and MFN2, that function in place of a single outer membrane DRP, Fzo1 in yeast. We addressed the significance of two mammalian outer membrane fusion DRPs using an in?vitro mammalian mitochondrial fusion assay. We demonstrate that heterotypic MFN1-MFN2 trans complexes possess greater efficacy in fusion as compared to homotypic MFN1 or MFN2 complexes. In addition, we show that the soluble form of the proapoptotic Bcl2 protein, Bax,?positively regulates mitochondrial fusion exclusively through homotypic MFN2 trans complexes. Together, these data demonstrate functional and regulatory distinctions between MFN1 and MFN2 and provide insight into their unique physiological roles.     

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-1β secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.     

MFN1在肝癌转移中的调控作用研究

目的:探讨线粒体融合蛋白MFN1(mito-fusion 1)在肝癌转移中的作用及其机制。方法:1).采用免疫组化实验检测15对肝癌转移灶组织与原发灶组织中MFN1的表达,以明确肝癌转移时是否伴有MFN1表达的改变。2).采用si RNA (small interference RNA)下调肝癌细胞中MFN1的表达后,提高Transwell迁移实验和Transwell侵袭实验分别检测其迁移和侵袭能力,通过实时荧光定量PCR (Quantitative Real-time PCR,qRT-PCR)和Western blot实验分别检测基质金属蛋白酶1 (matrix metalloproteinase 1,MMP1)、MMP2、MMP7及MMP9的m RNA和蛋白表达。结果:1)肝癌转移灶组织中MFN1表达显著低于原发灶组织(P0.05)。2).下调MFN1表达后,肝癌细胞的迁移和侵袭能力显著升高,MMP7的表达显著增加,而MMP1、MMP2与MMP9的表达无明显变化。结论:线粒体融合蛋白MFN1在肝癌转移组织中表达显著降低,可能通过激活MMP7表达,促进肝癌细胞侵袭和转移。     

A novel mutation of the MFN2 gene in a Chinese family with Charcot-Marie-Tooth disease

Charcot-Marie-Tooth (CMT) is a group of clinically and genetically heterogeneous inherited neuromuscular disorders. At present, more than 30 loci have been reported to be associated with CMT disease; point mutations in the mitofusin 2 (MFN2) gene is one of the most common causes. We studied a Chinese family with CMT disease in which the phenotype of affected individuals varied, and the weakness condition of the distal legs in males, except the proband, was less severe than in females in this family. Linkage analysis and PCR sequencing revealed a missense mutation (NM_014874.3:c.1066 A>G) in the MFN2 gene, resulting in an animo acid substitution of threonine to alanine in condon 356 (Thr356Ala). This is a novel phenotype and mutation for CMT family.     

MFN2 silencing promotes neural differentiation of embryonic stem cells via the Akt signaling pathway

Mitofusin 2 (MFN2) is a regulatory protein participating in mitochondria dynamics, cell proliferation, death, differentiation, and so on. This study aims at revealing the functional role of MFN2 in the pluripotency maintenance and primitive differetiation of embryonic stem cell (ESCs). A dox inducible silencing and routine overexpressing approach was used to downregulate and upregulate MFN2 expression, respectively. We have compared the morphology, cell proliferation, and expression level of pluripotent genes in various groups. We also used directed differentiation methods to test the differentiation capacity of various groups. The Akt signaling pathway was explored by the western blot assay. MFN2 upregulation in ESCs exhibited a typical cell morphology and similar cell proliferation, but decreased pluripotent gene markers. In addition, MFN2 overexpression inhibited ESCs differentiation into the mesendoderm, while MFN2 silencing ESCs exhibited a normal cell morphology, slower cell proliferation and elevated pluripotency markers. For differentiation, MFN2 silencing ESCs exhibited enhanced three germs' differentiation ability. Moreover, the protein levels of phosphorylated Akt308 and Akt473 decreased in MFN2 silenced ESCs, and recovered in the neural differentiation process. When treated with the Akt inhibitor, the neural differentiation capacity of the MFN2 silenced ESCs can reverse to a normal level. Taken together, the data indicated that the appropriate level of MFN2 expression is essential for pluripotency and differentiation capacity in ESCs. The increased neural differentiation ability by MFN2 silencing is strongly related to the Akt signaling pathway.     

When MFN2 (mitofusin 2) met autophagy: A new age for old muscles

A long-standing quest is to define the mechanisms responsible for the mitochondrial dysfunction and accumulation of damaged mitochondria that occur during aging. Indeed, those defects are considered major contributors to the aging process. We have analyzed the effect of aging on the muscle expression of Mfn2 and the impact of Mfn2 ablation on muscle function. Our findings reveal that Mfn2 is repressed in muscle during aging, and that is a determinant for the inhibition of autophagy, and mitochondrial quality control, which lead to the accumulation of damaged mitochondria.     

MFN2 gene analysis in patients with hereditary motor and sensory neuropathy from Bashkortostan Republic

Hereditary motor and sensory neuropathy (HMSN) type IIA is caused by mutations in the mitofusin type-2 (MFN2) gene and represents one of the most common axonal forms of HMSN. We determined the spectrum and frequency of MFN2 gene mutations in patients from the Bashkortostan Republic (BR). Four different mutations were revealed in 5 out of 170 unrelated patients, i.e., c.2113G>A (p.Val705Ile) (1.2% among all types of HMSN in the total sample of patients and 2% among patients of Tatar ethnicity). This mutation was described previously; c.775C>T (p.Arg259Cys) (0.6%, in the total sample of patients and 2% among the patients of Tatar ethnicity); c.776G>A (p.Arg259His) (0.6% in the total sample of patients and 1.5% among the patients of Russians ethnicity); and c.2171T>C (p.Leu724Pro) (1.2% in the total sample of patients and 7.4% among the patients of Bashkirs ethnicity). These are new mutations that were not observed among healthy family members and in control samples of healthy subjects. Five identified nucleotide substitutions represent single nucleotide polymorphisms of the gene, including c.892G>A (p.Gly298Arg), c.957C>T (Gly319Gly), and c1039-222t>c, which were described previously, while c.175+28c>t and c.2204+15t>c represent new nucleotide substitutions in the intron regions of the gene.     

Two de novo mutations of MFN2 associated with early-onset Charcot-Marie-Tooth disease type 2A neuropathy

Charcot-Marie-Tooth disease type 2A (CMT2A) is one of the subdivisions of CMT2, an axonal defective form of peripheral neuropathy. Different mutations in the mitochondrial GTPase mitofusin 2 (MFN2) gene produce various degrees of severity of CMT2A phenotype or CMT2A related hereditary motor and sensory neuropathy VI (HMSN VI). The occurrence of de novo mutations in MFN2 is by far the most frequent as compared to other CMT genes. About 26% of the pathogenic MFN2 mutations reported in the Inherited Peripheral Neuropathies Mutations Database are de novo. This study identified two de novo mutations of MFN2, c.1048T>C (S350P) and c.310C>T (R104W), from two Korean CMT2A patients with early onset severe clinical symptoms. The comparative genotype-phenotype correlations of these mutations according to a previously reported case were also viewed. The R104W mutation has been reported recurrently, outspread over different ethnic backgrounds as de novo. The re-occurrence of the same pathogenic de novo variants within and amongst different ethnic groups clearly suggests a susceptible hot spot for mutation in the MFN2 gene. If the deleterious mutations discourage fitness and reproduction, this negative selection factor should ultimately reduce the prevalence of the disease. It appears that spontaneous de novo mutations in turn seem to be maintaining the disease phenotype??s prevalence.     

Betaine enhances the cellular survival via mitochondrial fusion and fission factors,MFN2 and DRP1

Betaine is a key metabolite of the methionine cycle and known for attenuating alcoholic steatosis in the liver. Recent studies have focused on the protection effect of betaine in mitochondrial regulation through the enhanced oxidative phosphorylation system. However, the mechanisms of its beneficial effects have not been clearly identified yet. Mitochondrial dynamics is important for the maintenance of functional mitochondria and cell homeostasis. A defective mitochondrial dynamics and oxidative phosphorylation system have been closely linked to several pathologies, raising the possibility that novel drugs targeting mitochondrial dynamics may present a therapeutic potential to restore the cellular homeostasis. In this study, we investigated betaine’s effect on mitochondrial morphology and physiology and demonstrated that betaine enhances mitochondrial function by increasing mitochondrial fusion and improves cell survival. Furthermore, it rescued the unbalance of the mitochondrial dynamics from mitochondrial oxidative phosphorylation dysfunction induced by oligomycin and rotenone. The elongation properties by betaine were accompanied by lowering DRP1 and increasing MFN2 expression. These data suggest that betaine could play an important role in remodeling mitochondrial dynamics to enhance mitochondrial function and cell viability.     

MFN2-mediated mitochondrial fusion facilitates acute hypobaric hypoxia-induced cardiac dysfunction by increasing glucose catabolism and ROS production

BackgroundRapid ascent to high-altitude environment which is characterized by acute hypobaric hypoxia (HH) may increase the risk of cardiac dysfunction. However, the potential regulatory mechanisms and prevention strategies for acute HH-induced cardiac dysfunction have not been fully clarified. Mitofusin 2 (MFN2) is highly expressed in the heart and is involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the significance of MFN2 in the heart under acute HH has not been investigated.Methods and resultsOur study revealed that MFN2 upregulation in hearts of mice during acute HH led to cardiac dysfunction. In vitro experiments showed that the decrease in oxygen concentration induced upregulation of MFN2, impairing cardiomyocyte contractility and increasing the risk of QT prolongation. Additionally, acute HH-induced MFN2 upregulation promoted glucose catabolism and led to excessive mitochondrial reactive oxygen species (ROS) production in cardiomyocytes, ultimately resulting in decreased mitochondrial function. Furthermore, co-immunoprecipitation (co-IP) and mass spectrometry analyses indicated that MFN2 interacted with the NADH-ubiquinone oxidoreductase 23 kDa subunit (NDUFS8). Specifically, acute HH-induced MFN2 upregulation increased NDUFS8-dependent complex I activity.ConclusionsTaken together, our studies provide the first direct evidence that MFN2 upregulation exacerbates acute HH-induced cardiac dysfunction by increasing glucose catabolism and ROS production.General significanceOur studies indicate that MFN2 may be a promising therapeutic target for cardiac dysfunction under acute HH.     

MFN2-driven mitochondria-to-nucleus tethering allows a non-canonical nuclear entry pathway of the mitochondrial pyruvate dehydrogenase complex

1. Download : Download high-res image (254KB)
2. Download : Download full-size image

     

MFN1介导的线粒体融合在心肌细胞凋亡中的作用研究

目的:探讨线粒体融合关键蛋白MFN1介导的线粒体融合在调控心肌细胞凋亡中的作用。方法:通过si RNA降低体外培养H9C2心肌细胞中MFN1的表达后,采用Western blot检测线粒体细胞色素c(Cyto c)释放及其下游凋亡效应分子Caspase9与Caspase3活性,流式细胞术检测细胞内活性氧(ROS)的产生情况,流式细胞术检测细胞凋亡的情况。结果:干扰MFN1可显著促进H9C2心肌细胞内细胞色素c由线粒体释放至胞浆,促进Caspase9与Caspase3的激活,增加细胞内活性氧ROS产生并提高细胞凋亡率(均P0.05)。结论:MFN1介导的线粒体融合可保护心肌细胞凋亡,其机制可能与抑制ROS产生与细胞色素C释放有关。     

Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR_1–5) to regulate cellular events, including Ca²⁺ signaling. The SK1/S1P axis and Ca²⁺ signaling both play important roles in health and disease. In this respect, Ca²⁺ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca²⁺ release from the ER resulting in increased mitochondrial matrix Ca²⁺. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca²⁺ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca²⁺ and downstream cellular effects.     

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.     

Silibinin treatment results in reducing OPA1&MFN1 genes expression in a rat model hepatic ischemia–reperfusion

The mitochondrial damage has a pivotal role in triggering apoptosis and cell death. This study assessed the effect of silibinin on optical atrophy-1 (OPA1) and mitofusin-1 (MFN1) gene expression in liver tissue during hepatic warm ischemia–reperfusion (IR). Four groups of rats, eight rats each were designed: Vehicle: the rats received normal saline and encountered to laparotomy, Sili: silibinin (60 mg/kg) was administered to animals, IR: the rats received the normal saline and insulted by liver IR procedure, and IR?+?Sili: silibinin was injected to rats. All groups were subjected to the same process of injection of the solvent or silibinin (30 min before laparotomy or ischemia and immediately after the reperfusion), intraperitoneally (IP). After 3 h of reperfusion, blood and liver tissue samples were collected for future examinations. Our results showed no significant differences between the Vehicle and Sili groups in all assessed parameters. In IR?+?Sili, the increased serum levels of AST and ALT in comparison with the control group were markedly reduced by silibinin treatment. Silibinin lowered the elevated expression of OPA1 and MFN1 mRNAs in the IR group. Histology revealed silibinin could decline tissue degeneration compared to the IR group. Electron microscopy of control and silibinin groups showed no fusion of mitochondria and tissue degradation both of which were observed in the IR group. The extent of tissue destruction and mitochondrial fusion decreased significantly with silibinin treatment. Silibinin has a protective effect on liver cells against IR induced injuries by preserving mitochondrial membrane.