MFN2 Research Articles

Sporadic hereditary motor and sensory neuropathies: Advances in the diagnosis using next generation sequencing technology

Hereditary motor and sensory neuropathies (HMSN) are genetically heterogeneous disorders affecting peripheral motor and sensory functions. Many different pathogenic variants in several genes involved in the demyelinating, the axonal and the intermediate HMSN forms have been identified, for which all inheritance patterns have been described. The mutation screening currently available is based on Sanger sequencing and is time-consuming and relatively expensive due to the high number of genes involved and to the absence of mutational hot spots. To overcome these limitations, we have designed a custom panel for simultaneous sequencing of 28 HMSN-related genes. We have applied this panel to three representative patients with variable HMSN phenotype and uncertain diagnostic classifications. Using our NGS platform we rapidly identified three already described pathogenic heterozygous variants in MFN2, MPZ and DNM2 genes. Here we show that our pre-custom platform allows a fast, specific and low-cost diagnosis in sporadic HMSN cases. This prompt diagnosis is useful for providing a well-timed treatment, establishing a recurrence risk and preventing further investigations poorly tolerated by patients and expensive for the health system. Importantly, our study illustrates the utility and successful application of NGS to mutation screening of a Mendelian disorder with extreme locus heterogeneity.

Clinical and genetic spectra in a series of Chinese patients with Charcot–Marie–Tooth disease

The aim of this study was to determine the clinical features and frequencies of genetic subtypes in a series of patients with Charcot-Marie-Tooth (CMT) disease from Eastern China. Patients were divided into three subtypes, CMT1, CMT2 and hereditary neuropathy with liability to pressure palsy (HNPP), according to their electrophysiological manifestations. Multiplex ligation-dependent probe analysis (MLPA) was performed to detect duplications/deletions in the PMP22 gene. The coding regions and splice sites of the GJB1, MPZ, MFN2 and GDAP-1 genes were determined by direct sequencing. Among the 148 patients in the study, 37.2% of the cases had mutations in genes assessed. The mutation detection rate was higher in patients with family histories than in spontaneous cases. PMP22 duplication (13.5%) was predominant in this group of patients, followed by PMP22 deletion (11.5%), and point mutations in GJB1 (8.8%), MPZ (2.0%) and MFN2 (0.7%). Three novel mutations (c.151T>C and c.310 A>G in GJB1 and c.1516 C>G in MFN2) were detected. A small deletion in PMP22 exon 4 was detected in a patient with severe CMT1. Genetic tests have great value in CMT patients with family histories. The frequency of PMP22 duplications was lower in Asian patients than in others. We suggest that genetic testing strategies in CMT patients should be primarily based on electromyography data.

Pro-apoptotic and anti-proliferative effects of mitofusin-2 via PI3K/Akt signaling in breast cancer cells.

The mitochondrial GTPase mitofusin-2 (Mfn2) gene is a novel gene characterized as a cell proliferation inhibitor. Mfn2 has previously been reported to play a role in regulating cell proliferation, apoptosis and differentiation in a number of cell types. However, there are no studies on the effect of Mfn2 in breast cancer. In this study, we aimed to elucidate the function and mechanism of Mfn2 in breast cancer. A plasmid encoding the complete Mfn2 open reading frame (pEGFP-Mfn2) was used to infect breast cancer cells. The effect of Mfn2 on proliferation was assessed by methyl thiazolyl tetrazolium and bromodeoxyuridine incorporation analyses. Flow cytometry, immunofluorescence and western blot analyses were used to test the effects of Mfn2 on cell cycle distribution and apoptosis. Additionally, the PI3K/Akt signaling pathway was analyzed after pEGFP-Mfn2 was transfected into MCF-7 cells. The results revealed that Mfn2 suppressed the proliferation of MCF-7 cells by regulating more cells at the G0/G1 phase and decreasing proliferating cell nuclear antigen and cyclin A expression. The results also demonstrated that the PI3K/Akt signaling pathway is involved in Mfn2-regulated proliferation and apoptosis. Taken together, this indicates that Mfn2 mediates MCF-7 cell proliferation and apoptosis via the PI3K/Akt signaling pathway. Mfn2 may thus be a significant therapeutic target in the treatment of breast cancer.

Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells.

Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism.

Mfn2 Affects Embryo Development via Mitochondrial Dysfunction and Apoptosis.

BackgroundGrowth factors, energy sources, and mitochondrial function strongly affect embryo growth and development in vitro. The biological role and prospective significance of the mitofusin gene Mfn2 in the development of preimplantation embryos remain poorly understood. Our goal is to profile the role of Mfn2 in mouse embryos and determine the underlying mechanism of Mfn2 function in embryo development.MethodsWe transfected Mfn2-siRNA into 2-cell fertilized eggs and then examined the expression of Mfn2, the anti-apoptotic protein Bcl-2, and the apoptosis-promoting protein Bax by Western blot. Additionally, we determined the blastocyst formation rate and measured ATP levels, mtDNA levels, mitochondrial membrane potential (ΔΨm), and apoptosis in all of the embryos.ResultsThe results indicate that the Mfn2 and Bcl-2 levels were markedly decreased, whereas Bax levels were increased in the T group (embryos transfected with Mfn2-siRNA) compared with the C group (embryos transfected with control-siRNA). The blastocyst formation rate was significantly decreased in the T group. The ATP content and the relative amounts of mtDNA and cDNA in the T group were significantly reduced compared with the C group. In the T group, ΔΨm and Ca2+ levels were reduced, and the number of apoptotic cells was increased.ConclusionLow in vitro expression of Mfn2 attenuates the blastocyst formation rate and cleavage speed in mouse zygotes and causes mitochondrial dysfunction, as confirmed by the ATP and mtDNA levels and mitochondrial membrane potential. Mfn2 deficiency induced apoptosis through the Bcl-2/Bax and Ca2+ pathways. These findings indicate that Mfn2 could affect preimplantation embryo development through mitochondrial function and cellular apoptosis.

Mitofusin 2 is required to maintain mitochondrial coenzyme Q levels.

Mitochondria form a dynamic network within the cell as a result of balanced fusion and fission. Despite the established role of mitofusins (MFN1 and MFN2) in mitochondrial fusion, only MFN2 has been associated with metabolic and neurodegenerative diseases, which suggests that MFN2 is needed to maintain mitochondrial energy metabolism. The molecular basis for the mitochondrial dysfunction encountered in the absence of MFN2 is not understood. Here we show that loss of MFN2 leads to impaired mitochondrial respiration and reduced ATP production, and that this defective oxidative phosphorylation process unexpectedly originates from a depletion of the mitochondrial coenzyme Q pool. Our study unravels an unexpected and novel role for MFN2 in maintenance of the terpenoid biosynthesis pathway, which is necessary for mitochondrial coenzyme Q biosynthesis. The reduced respiratory chain function in cells lacking MFN2 can be partially rescued by coenzyme Q10 supplementation, which suggests a possible therapeutic strategy for patients with diseases caused by mutations in the Mfn2 gene.

Improved inherited peripheral neuropathy genetic diagnosis by whole-exome sequencing.

Inherited peripheral neuropathies (IPNs) are a group of related diseases primarily affecting the peripheral motor and sensory neurons. They include the hereditary sensory neuropathies (HSN), hereditary motor neuropathies (HMN), and Charcot-Marie-Tooth disease (CMT). Using whole-exome sequencing (WES) to achieve a genetic diagnosis is particularly suited to IPNs, where over 80 genes are involved with weak genotype–phenotype correlations beyond the most common genes. We performed WES for 110 index patients with IPN where the genetic cause was undetermined after previous screening for mutations in common genes selected by phenotype and mode of inheritance. We identified 41 missense sequence variants in the known IPN genes in our cohort of 110 index patients. Nine variants (8%), identified in the genes MFN2, GJB1, BSCL2, and SETX, are previously reported mutations and considered to be pathogenic in these families. Twelve novel variants (11%) in the genes NEFL, TRPV4, KIF1B, BICD2, and SETX are implicated in the disease but require further evidence of pathogenicity. The remaining 20 variants were confirmed as polymorphisms (not causing the disease) and are detailed here to help interpret sequence variants identified in other family studies. Validation using segregation, normal controls, and bioinformatics tools was valuable as supporting evidence for sequence variants implicated in disease. In addition, we identified one SETX sequence variant (c.7640T>C), previously reported as a putative mutation, which we have confirmed as a nonpathogenic rare polymorphism. This study highlights the advantage of using WES for genetic diagnosis in highly heterogeneous diseases such as IPNs and has been particularly powerful in this cohort where genetic diagnosis could not be achieved due to phenotype and mode of inheritance not being previously obvious. However, first tier testing for common genes in clinically well-defined cases remains important and will account for most positive results.

25. A new mutation for asymmetrical inherited neuropathy: The help of ultrasound study in a paediatric diagnostic challenge

Paediatric neuromuscular diseases represent a diagnostic challenge often requiring invasive investigations hardly tolerate by children. We describe the case of a young athlet, male,13 years-old, presenting a subacute progressive right pes-cavus and lower limb’s hypometria. This clinical presentation was noted following an increase in height of child and also in correspondence of an accidental crash. Initially his deficit was setted as traumatic but further clinical development proposed again the diagnostic doubt. A neuromuscular-ultrasound(NMUS)-study showed a bilateral involvement of lower limb?s posterior loggias, mainly right, with echogenicity homogeneously increased and preserved architecture. The EMG-study showed a distal asymmetric polyneuropathy, predominant in the Tibial nerve. Similar EMG-results were found in his father. No encephalic-RMI alterations. In his parents, the immunological investigations resulted normal while the genetic ones showed a novel heterozygosis mutation of MFN2 gene. Further NMUS-studies were performed to follow up. This paucisymptomatic, monolateral, late-onset clinical case showed how a preliminary NMUS-study can guide further investigations. A prevalent chronic damage with muscular structure preserved and EMG-detection of asymmetric-neuropathy could exclude a traumatic genesis guiding diagnosis to an inherited asymmetrical neuropathy. The combined morpho-functional evaluation with integrated NMUS-EMG facilitates pediatric neuromuscular diagnosis, improving spatial resolution with limited costs and invasivness.

Mitochondrial remodeling in mice with cardiomyocyte-specific lipid overload

BackgroundObesity leads to metabolic heart disease (MHD) that is associated with a pathologic increase in myocardial fatty acid (FA) uptake and impairment of mitochondrial function. The mechanism of mitochondrial dysfunction in MHD, which results in oxidant production and decreased energetics, is poorly understood but may be related to excess FAs. Determining the effects of cardiac FA excess on mitochondria can be hindered by the systemic sequelae of obesity. Mice with cardiomyocyte-specific overexpression of the fatty acid transport protein FATP1 have increased cardiomyocyte FA uptake and develop MHD in the absence of systemic lipotoxicity, obesity or diabetes. We utilized this model to assess 1) the effect of cardiomyocyte lipid accumulation on mitochondrial structure and energetic function and 2) the role of lipid-driven transcriptional regulation, signaling, toxic metabolite accumulation, and mitochondrial oxidative stress in lipid-induced MHD. MethodsCardiac lipid species, lipid-dependent signaling, and mitochondrial structure/function were examined from FATP1 mice. Cardiac structure and function were assessed in mice overexpressing both FATP1 and mitochondrial-targeted catalase. ResultsFATP1 hearts exhibited a net increase (+12%) in diacylglycerol, with increases in several very long-chain diacylglycerol species (+160–212%, p<0.001) and no change in ceramide, sphingomyelin, or acylcarnitine content. This was associated with an increase in phosphorylation of PKCα and PKCδ, and a decrease in phosphorylation of AKT and expression of CREB, PGC1α, PPARα and the mitochondrial fusion genes MFN1, MFN2 and OPA1. FATP1 overexpression also led to marked decreases in mitochondrial size (−49%, p<0.01), complex II-driven respiration (−28.6%, p<0.05), activity of isolated complex II (−62%, p=0.05), and expression of complex II subunit B (SDHB) (−60% and −31%, p<0.01) in the absence of change in ATP synthesis. Hydrogen peroxide production was not increased in FATP1 mitochondria, and cardiac hypertrophy and diastolic dysfunction were not attenuated by overexpression of catalase in mitochondria in FATP1 mice. ConclusionsExcessive delivery of FAs to the cardiac myocyte in the absence of systemic disorders leads to activation of lipid-driven signaling and remodeling of mitochondrial structure and function.

G.P.296: Severe, early onset Charcot-Marie-Tooth disease with rare presentations

Charcot–Marie–Tooth disease (CMT) has diverse clinical presentations and molecular genetic heterogeneity. Herein we describe the clinical features, neurophysiological and morphological studies of 2 fatal cases of CMT. Case 1 was a healthy newborn who developed foot drop, daily frequent vomiting and severe constipation at 7 months of age. His arm and leg weakness rapidly progressed within a few months. He subsequently developed diaphragmatic paralysis and died at 2.5 years of age due to respiratory failure. NCS/EMG showed a severe, axonal sensory and motor neuropathy. He had a novel and de novo mutation (c.296–297dupAAG) of the MFN2 gene. Case 2 had congenital hypomyelinating neuropathy confirmed by NCS and nerve biopsy, associated with a novel and de novo missense mutation (p.His384Arg) of the EGR2 gene. The patient presented a few weeks after birth with failure to thrive and hypotonia. She had facial, severe proximal > distal arm and leg weakness, pain insensitivity, areflexia and symptoms of autonomic dysfunction. She progressed to respiratory failure requiring mechanical ventilation. Her CSF protein was elevated (136 mg/dl). She had a fluctuating clinical course that let to the initial consideration of chronic inflammatory demyelinting polyneuropathy. She did not respond to prednisone, but she responded to IVIG therapy initially with some improvement of muscle strength and decrease autonomic dysfunction. She subsequently experienced deterioration of weakness not responding to either IVIG or plasmapheresis, and died at 16.5 months of age. Our report expanded the genotype and phenotype of MFN2 and EGR2 mutations. Although early onset CMT has been well described, autonomic features and fatality due to respiratory failure are rarely described. MFN2 mutations frequently cause severe phenotype, but diaphragmatic paralysis and autonomic dysfunction have not been described. Similarly, severe autonomic dysfunction has not been reported to be associated with EGR2 mutations.

The allelic spectrum of Charcot-Marie-Tooth disease in over 17,000 individuals with neuropathy.

We report the frequency, positive rate, and type of mutations in 14 genes (PMP22, GJB1, MPZ, MFN2, SH3TC2, GDAP1, NEFL, LITAF, GARS, HSPB1, FIG4, EGR2, PRX, and RAB7A) associated with Charcot–Marie–Tooth disease (CMT) in a cohort of 17,880 individuals referred to a commercial genetic testing laboratory. Deidentified results from sequencing assays and multiplex ligation-dependent probe amplification (MLPA) were analyzed including 100,102 Sanger sequencing, 2338 next-generation sequencing (NGS), and 21,990 MLPA assays. Genetic abnormalities were identified in 18.5% (n = 3312) of all individuals. Testing by Sanger and MLPA (n = 3216) showed that duplications (dup) (56.7%) or deletions (del) (21.9%) in the PMP22 gene accounted for the majority of positive findings followed by mutations in the GJB1 (6.7%), MPZ (5.3%), and MFN2 (4.3%) genes. GJB1 del and mutations in the remaining genes explained 5.3% of the abnormalities. Pathogenic mutations were distributed as follows: missense (70.6%), nonsense (14.3%), frameshift (8.7%), splicing (3.3%), in-frame deletions/insertions (1.8%), initiator methionine mutations (0.8%), and nonstop changes (0.5%). Mutation frequencies, positive rates, and the types of mutations were similar between tests performed by either Sanger (n = 17,377) or NGS (n = 503). Among patients with a positive genetic finding in a CMT-related gene, 94.9% were positive in one of four genes (PMP22, GJB1, MPZ, or MFN2).

Charcot-Marie-Tooth disease type 2A: from typical to rare phenotypic and genotypic features.

Axonal Charcot-Marie-Tooth disease (CMT) is genetically heterogeneous, with 11 genes identified. Axonal CMT has most frequently been associated with mutations in the MFN2 gene (CMT2A). To describe the clinical and molecular features of CMT2A, to delineate prognostic factors, to understand connections between a certain phenotype and more serious clinical consequences, and to identify interactions among the associated genes. We describe the clinical, molecular, electrophysiological, and additional features of 43 patients with CMT2A. The degree of physical disability was determined by the CMT neuropathy score and adapted to the CMT neuropathy score gradient to evaluate the clinical course. We evaluated all data within the context of the most recent and important publications concerning this issue. Twenty-five patients had early-onset CMT2A and severe functional disability, with 9 being wheelchair bound, and 18 had late-onset disease and a milder phenotype. Optic atrophy, vocal cord palsy, and auditory impairment were observed in 5, 6, and 2 patients, respectively. Among the 24 patients who underwent magnetic resonance imaging of the spinal cord, 6 had evidence of spinal atrophy with or without hydromyelia. In 1 patient, magnetic resonance imaging revealed hydrocephalus. Twenty different MFN2 mutations were identified, and 14 were considered new variants. Their transmission was predominantly autosomal dominant, with vertical transmission in 8 and de novo occurrence in 3. However, we also identified rare types of transmission, especially a germinal mosaicism and an autosomal recessive inheritance. One patient carried a rare variant in the GDAP1 gene and another in the OPA1 gene in association with MFN2 mutation. Charcot-Marie-Tooth disease type 2A associated with MFN2 mutations is clinically very heterogeneous. Ranging from a mild to a severe form, CMT2A exhibits various types of transmission. Optic atrophy and vocal cord palsy were observed in patients with severe disability and an early-onset form and also in patients with later onset. Hydromyelia and spinal cord atrophy support central nervous system involvement in CMT2A.

Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro

Astrocytes become activated in response to central nervous system (CNS) injury, and excessive astrogliosis is considered an impediment to axonal regeneration by forming glial scar. Mitofusin 2 (Mfn2), a key protein in mitochondrial network, has been reported to negatively regulate cell proliferation. The present study aimed to explore whether reactive astrogliosis could be suppressed by Mfn2 overexpression. Scratch injury and starvation-serum stimulation models in cultured astrocytes were combined to address this issue. In scratch model, reactive proliferation status of damaged astrocytes was implicated by migration of high ratio of EdU(+) cells into lesion region and significantly increased expression of GFAP and PCNA. At meantime, Mfn2 expression was found to exert a down-regulated trend both in gen and protein levels. Pretreatment of cells with adenoviral vector encoding Mfn2 gene increased Mfn2 expression and subsequently attenuated injury-induced astrocytes hyperplasia, activation-relevant protein synthesis, cellular proliferation, eventually delayed wound healing process. Furthermore, Mfn2 overexpression markedly inhibited astrocytes proliferation induced by serum stimulation, by arresting the transition of cell cycle from G1 to S phase. Together, these in vitro results demonstrated that reactive astrogliosis can be effectively suppressed by up-regulation of Mfn2, which might contribute to a promising therapeutic intervention in CNS disease characterized by glia-related damage.

Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2

Previous studies have shown that epigallocatechin-3-gallate (EGCG) inhibits the proliferation of vascular smooth muscle cells (VSMCs) via the extracellular-signal-regulated kinase (ERK1/2) and mitogen activated protein kinases (MAPKs) pathway. Mitofusin 2 (Mfn-2) also suppresses VSMC proliferation through Ras-Raf-ERK/MAPK, suggesting a possible link between EGCG, Mfn-2 and ERK/MAPK. However, the effect of EGCG on Mfn-2 remains unknown. In this study, we investigated the role of Mfn-2 in the regulation of VSMC proliferation by EGCG, and assessed the underlying mechanisms. The effects of EGCG on the proliferation of cultured human aortic smooth muscle cells (HASMCs) were observed by 5-ethynl-2-deoxyuridine (EdU) incorporation assay. Mfn-2 gene and protein levels, and Ras, p-c-Raf and p-ERK1/2 protein levels were determined by quantitative real-time polymerase chain reaction and western blotting, respectively. Mfn-2 gene silencing was achieved by RNA interference. EGCG 50μmol/L profoundly inhibited the proliferation of HASMCs in culture, up-regulated Mfn-2, and down-regulated the expression of p-c-Raf and p-ERK1/2. Furthermore, RNA interference-mediated gene knockdown of Mfn-2 antagonized EGCG-induced anti-proliferation and down-regulation of Ras, p-c-Raf and p-ERK1/2. These results suggest that EGCG inhibits the proliferation of HASMCs in vitro largely via Mfn-2-mediated suppression of the Ras-Raf-ERK/MAPK signaling pathway.

Epigallocatechin gallate counteracts oxidative stress in docosahexaenoxic acid-treated myocytes

Skeletal muscle is a key organ of mammalian energy metabolism, and its mitochondria are multifunction organelles that are targets of dietary bioactive compounds. The goal of this work was to examine the regulation of mitochondrial dynamics, functionality and cell energy parameters using docosahexaenoic acid (DHA), epigallocatechin gallate (EGCG) and a combination of both in L6 myocytes. Compounds (at 25μM) were incubated for 4h. Cells cultured with DHA displayed less oxygen consumption with higher ADP/ATP ratio levels concomitant with downregulation of Cox and Ant1 gene expression. The disruption of energetic homeostasis by DHA, increases intracellular reactive oxygen species (ROS) levels and decreases mitochondrial membrane potential. The defence mechanism to counteract the excess of ROS production was by the upregulation of Ucp2, Ucp3 and MnSod gene expression. Moreover myocytes cultured with DHA had a higher mitochondrial mass with a higher proportion of large and elongated mitochondria, whereas the fission genes Drp1 and Fiss1 and the fusion gene Mfn2 were downregulated. In myocytes co-incubated with DHA and EGCG, ROS levels and the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio were similar to untreated myocytes and the decrease of oxygen consumption, higher mitochondrial mass and the overexpression of Ucp2 and Ucp3 genes were similar to the DHA-treated cells with also a higher amount of mitochondrial deoxyribonucleic acid (DNA), and reduced Drp1 and Fiss1 gene expression levels. In conclusion the addition of EGCG to DHA returned the cells to the control conditions in terms of mitochondrial morphology, energy and redox status, which were unbalanced in the DHA-treated myocytes.

Mitofusin 2 expression dominates over mitofusin 1 exclusively in mouse dorsal root ganglia - a possible explanation for peripheral nervous system involvement in Charcot-Marie-Tooth 2A.

Mitofusin 2 (Mfn2), a protein of the mitochondrial outer membrane, is essential for mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. Mutations in the mitofusin 2 gene cause axonal Charcot-Marie-Tooth type 2A (CMT2A), an inherited disease affecting peripheral nerve axons. The precise mechanism by which mutations in MFN2 selectively cause the degeneration of long peripheral axons is not known. There is a hypothesis suggesting the involvement of reduced expression of a homologous protein, mitofusin 1 (Mfn1), in the peripheral nervous system, and less effective compensation of defective mitofusin 2 by mitofusin 1. We therefore aimed to perform an analysis of the mitofusin 1 and mitofusin 2 mRNA and protein expression profiles in different mouse tissues, with special attention paid to dorsal root ganglia (DRGs), as parts of the peripheral nervous system. Quantitative measurement relating to mRNA revealed that expression of the Mfn2 gene dominates over Mfn1 mainly in mouse DRG, as opposed to other nervous system samples and other tissues studied. This result was further supported by Western blot evaluation. Both these sets of data confirm the hypothesis that the cellular consequences of mutations in the mitofusin 2 gene can mostly be manifested in the peripheral nervous system.

MicroRNA-19b targets Mfn1 to inhibit Mfn1-induced apoptosis in osteosarcoma cells.

Accumulative evidence has confirmed that, miR-17-92, a typical polycistronic mRNA cluster, was up-regulated in various solid tumors, and play an important role in the occurrence and development progress of tumors. In our study, we detected the six members of miR-17-92 cluster in osteosarcoma cell line, finding that the expression of miR-17 and miR-19b was up-regulated significantly. Further studies have found that Mfn1 was one of the target genes of miR-19b and the transcription and expression level of Mfn1 were down-regulated by miR-19b. MTS, flow cytometry, TUNEL-DAPI, Annexin V-FITC and transwell assay demonstrated that Mfn1 significantly blocked the cell cycle, promoted apoptosis and inhibited proliferation and invasion of osteosarcoma cells. Whereas, miR-19b targets 3'UTR sequences of Mfn1 genes inhibit the expression of Mfn1, thus inhibited Mfn1 triggered anti-cancer effect. Taken together, miR-19b functions by targeting Mfn1 reduce the protein expression level, thus provides a novel target to understand the molecular biology and genetics mechanisms of occurrence and development of osteosarcoma, contributing to the diagnosis and therapy of osteosarcoma.

MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age

To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.

Mitochondrial Genome Linearization Is a Causative Factor for Cardiomyopathy in Mice and Drosophila

Mitofusin (Mfn)2 redundantly promotes mitochondrial outer membrane tethering and organelle fusion with Mfn1, and uniquely functions as the mitochondrial receptor for Parkin during PTEN-induced putative kinase 1 (PINK1)-Parkin-mediated mitophagy. Selective deletion of Mfn2 with retention of Mfn1 preserves mitochondrial fusion while rendering damaged mitochondria resistant to normal quality control culling mechanisms. Consequently, neuron and cardiomyocyte-specific Mfn2 gene ablation is associated with accumulation of damaged mitochondria and organ dysfunction. Here, we determined how mitochondrial DNA (mtDNA) damage contributes to cardiomyopathy in Mfn2-deficient hearts. RNA sequencing of Mfn2-deficient hearts revealed increased expression of some nuclear-encoded mitochondrial genes, but mitochondrial-encoded transcripts were not upregulated in parallel and mtDNA content was decreased. Ultra-deep sequencing of mtDNA showed no increase in single nucleotide mutations, but copy number variations representing insertion-deletion (in-del) mutations were induced over time by cardiomyocyte-specific Mfn2 deficiency. Double-strand mtDNA breaks in the form of in-dels were confirmed by polymerase chain reaction, and in the form of linear mitochondrial genomes were identified by southern blot analysis. Linearization of Drosophila cardiomyocyte mtDNA using conditional cardiomyocyte-specific expression of mitochondrial targeted XhoI recapitulated the cardiomyopathy of Mfn2-deficient mouse hearts. This is the first description of mitochondrial genome linearization as a causative factor in cardiomyopathy. One of the consequences of interrupting mitochondrial culling by the PINK1-Mfn2-Parkin mechanism is an increase in mtDNA double-stranded breaks, which adversely impact mitochondrial function and DNA replication.

Reduced VDAC1 Protects Against Alzheimer's Disease, Mitochondria, and Synaptic Deficiencies

The objective of this study was to elucidate the effect of VDAC1 on Alzheimer's disease (AD)-related genes, mitochondrial activity, and synaptic viability. Recent knockout studies of VDAC1 revealed that homozygote VDAC1 knockout (VDAC1-/-) mice exhibited disrupted learning and synaptic plasticity, and in contrast, VDAC1+/- mice appeared normal in terms of lifespan, fertility, and viability relative to wild-type mice. However, the effects of reduced VDAC1 on mitochondrial/synaptic genes and mitochondrial function in AD-affected neurons are not well understood. In the present study, we characterized mitochondrial/synaptic and AD-related genes and mitochondrial function in VDAC1+/- mice and VDAC1+/+ mice. We found reduced mRNA levels in the AD-related genes, including AβPP, Tau, PS1, PS2, and BACE1; increased levels of the mitochondrial fusion genes Mfn1, Mfn2; reduced levels of the fission genes Drp1 and Fis1; and reduced levels of the mitochondrial permeability transition pore genes VDAC1, ANT, and CypD in VDAC1+/- mice relative to VDAC1+/+ mice. Hexokinase 1 and 2 were significantly upregulated in the VDAC+/- mice. The synaptic genes synaptophysin, synapsin 1 and 2, synaptobrevin 1 and 2, neurogranin, and PSD95 were also upregulated in the VDAC1+/- mice. Free radical production and lipid peroxidation levels were reduced in the VDAC1+/- mice, and cytochrome oxidase activity and ATP levels were elevated, indicating enhanced mitochondrial function in the VDAC1+/- mice. These findings suggest that reduced VDAC1 expression, such as that we found in the VDAC1+/- mice, may be beneficial to synaptic activity, may improve function, and may protect against toxicities of AD-related genes.