The discovery of the multiple roles of mitochondria-endoplasmic reticulum (ER) juxtaposition in cell biology often relied upon the exploitation of Mitofusin (Mfn) 2 as an ER-mitochondria tether. However, this established Mfn2 function was recently questioned, calling for a critical re-evaluation of Mfn2's role in ER-mitochondria cross-talk. Electron microscopy and fluorescence-based probes of organelle proximity confirmed that ER-mitochondria juxtaposition was reduced by constitutive or acute Mfn2 deletion. Functionally, mitochondrial uptake of Ca 2+ released from the ER was reduced following acute Mfn2 ablation, as well as in Mfn2−/− cells overexpressing the mitochondrial calcium uniporter. Mitochondrial Ca 2+ uptake rate and extent were normal in isolated Mfn2 −/− liver mitochondria, consistent with the finding that acute or chronic Mfn2 ablation or overexpression did not alter mitochondrial calcium uniporter complex component levels. Hence, Mfn2 stands as a bona fide ER-mitochondria tether whose ablation decreases interorganellar juxtaposition and communication.he endoplasmic reticulum (ER) and mitochondria are physically coupled to control mitochondrial Ca 2+ uptake, lipid transfer, autophagosome formation, ER stress, and apoptosis (1-6). Juxtaposition is mediated by protein structures that can be visualized in electron microscopy (EM) and electron tomography (ET) studies. These physical tethers span 6-15 nm when connecting smooth, and 19-30 nm when connecting rough ER to mitochondria (7). Operationally, an ER-mitochondria tether should fulfill at least these minimal criteria: (i) it is retrieved on the outer mitochondrial membrane (OMM); (ii) it is retrieved in mitochondria-associated ER membranes, the ER subdomain involved in interaction with mitochondria; (iii) it interacts in trans with a homo-or heterotypic interactor on the opposing membrane; (iv) its deletion increases the distance between the ER and mitochondria; or (v) its deletion reduces exchange of Ca 2+ and lipids between the ER and mitochondria. The molecular nature of ER-mitochondria tethers remained elusive for many years. The scaffold protein PACS2 (phosphofurin acidic cluster sorting protein 2) modulates their extent (8), and they include the heterotypic association between the inositol triphosphate (IP3) receptor on the ER and the OMM voltagedependent anion channel (9). Another protein that fulfils the operational criteria to be defined as a tether is Mitofusin 2 (Mfn2). This OMM profusion protein is also retrieved in mitochondria-associated ER membranes and ER Mfn2 interacts in trans with Mfn1 or Mfn2 on the mitochondria to physically tether the organelles. Mfn2 ablation increases the distance between the ER and mitochondria and decreases agonist-evoked Ca 2+ transfer from the ER to mitochondria (10) that depends on the generation of high Ca 2+ microdomains at their interface (11,12). The role of Mfn2 as a tether was confirmed independently in the heart (13), in pro-opiomelanocortin neurons (14), and in the liver (15).Mfn2-dependent tethe...

The primary gene mutated in Charcot-Marie-Tooth type 2A is mitofusin-2 (Mfn2). Mfn2 encodes a mitochondrial protein that participates in the maintenance of the mitochondrial network and that regulates mitochondrial metabolism and intracellular signaling. The potential for regulation of human Mfn2 gene expression in vivo is largely unknown. Based on the presence of mitochondrial dysfunction in insulin-resistant conditions, we have examined whether Mfn2 expression is dysregulated in skeletal muscle from obese or nonobese type 2 diabetic subjects, whether muscle Mfn2 expression is regulated by body weight loss, and the potential regulatory role of tumor necrosis factor (TNF)␣ or interleukin-6. We show that mRNA concentration of Mfn2 is decreased in skeletal muscle from both male and female obese subjects. Muscle Mfn2 expression was also reduced in lean or in obese type 2 diabetic patients. There was a strong negative correlation between the Mfn2 expression and the BMI in nondiabetic and type 2 diabetic subjects. A positive correlation between the Mfn2 expression and the insulin sensitivity was also detected in nondiabetic and type 2 diabetic subjects. To determine the effect of weight loss on Mfn2 mRNA expression, six morbidly obese subjects were subjected to weight loss by bilio-pancreatic diversion. Mean expression of muscle Mfn2 mRNA increased threefold after reduction in body weight, and a positive correlation between muscle Mfn2 expression and insulin sensitivity was again detected. In vitro experiments revealed an inhibitory effect of TNF␣ or interleukin-6 on Mfn2 expression in cultured cells. We conclude that body weight loss upregulates the expression of Mfn2 mRNA in skeletal muscle of obese humans, type 2 diabetes downregulates the expression of Mfn2 mRNA in skeletal muscle, Mfn2 expression in skeletal muscle is directly proportional to insulin sensitivity and is inversely proportional to the BMI, TNF␣ and interleukin-6 downregulate Mfn2 expression and may participate in the dysregulation of Mfn2 expression in obesity or type 2 diabetes, and the in vivo modulation of Mfn2 mRNA levels is an additional level of regulation for the control of muscle metabolism and could provide a molecular mechanism for alterations in mitochondrial function in obesity or type 2 diabetes. Diabetes 54: [2685][2686][2687][2688][2689][2690][2691][2692][2693] 2005

OBJECTIVEType 2 diabetes is associated with insulin resistance and skeletal muscle mitochondrial dysfunction. We have found that subjects with early-onset type 2 diabetes show incapacity to increase Vo2max in response to chronic exercise. This suggests a defect in muscle mitochondrial response to exercise. Here, we have explored the nature of the mechanisms involved.RESEARCH DESIGN AND METHODSMuscle biopsies were collected from young type 2 diabetic subjects and obese control subjects before and after acute or chronic exercise protocols, and the expression of genes and/or proteins relevant to mitochondrial function was measured. In particular, the regulatory pathway peroxisome proliferator–activated receptor γ coactivator (PGC)-1α/mitofusin-2 (Mfn2) was analyzed.RESULTSAt baseline, subjects with diabetes showed reduced expression (by 26%) of the mitochondrial fusion protein Mfn2 and a 39% reduction of the α-subunit of ATP synthase. Porin expression was unchanged, consistent with normal mitochondrial mass. Chronic exercise led to a 2.8-fold increase in Mfn2, as well as increases in porin, and the α-subunit of ATP synthase in muscle from control subjects. However, Mfn2 was unchanged after chronic exercise in individuals with diabetes, whereas porin and α-subunit of ATP synthase were increased. Acute exercise caused a fourfold increase in PGC-1α expression in muscle from control subjects but not in subjects with diabetes.CONCLUSIONSOur results demonstrate alterations in the regulatory pathway that controls PGC-1α expression and induction of Mfn2 in muscle from patients with early-onset type 2 diabetes. Patients with early-onset type 2 diabetes display abnormalities in the exercise-dependent pathway that regulates the expression of PGC-1α and Mfn2.

The interface between mitochondria and the endoplasmic reticulum is emerging as a crucial hub for calcium signalling, apoptosis, autophagy and lipid biosynthesis, with far reaching implications in cell life and death and in the regulation of mitochondrial and endoplasmic reticulum function. Here we review our current knowledge on the structural and functional aspects of this interorganellar juxtaposition. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Trichoplein/mitostatin (TpMs) is a keratin-binding protein that partly colocalizes with mitochondria and is often downregulated in epithelial cancers, but its function remains unclear. In this study, we report that TpMs regulates the tethering between mitochondria and endoplasmic reticulum (ER) in a Mitofusin 2 (Mfn2)-dependent manner. Subcellular fractionation and immunostaining show that TpMs is present at the interface between mitochondria and ER. The expression of TpMs leads to mitochondrial fragmentation and loosens tethering with ER, whereas its silencing has opposite effects. Functionally, the reduced tethering by TpMs inhibits apoptosis by Ca 2+ -dependent stimuli that require ER-mitochondria juxtaposition. Biochemical and genetic evidence support a model in which TpMs requires Mfn2 to modulate mitochondrial shape and tethering. Thus, TpMs is a new regulator of mitochondria-ER juxtaposition.

In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5’ AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans , deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1 -deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.

Aims/hypothesis: In obesity the cellular capacity to switch from using lipid to carbohydrate and vice versa as the energy substrate, known as 'metabolic flexibility', is impaired. Mitofusin 2 (MFN2), a mitochondrial membrane protein, seems to contribute to the maintenance and operation of the mitochondrial network, and its expression is reduced in obesity. The aim of this study was to verify whether MFN2 might be implicated in the metabolic inflexibility of obesity. Materials and methods: Insulin sensitivity was measured in six morbidly obese women before and 2 years after malabsorptive bariatric surgery (BMI 53.3±10.5 vs 30.3±4.0 kg/m 2 ). Skeletal muscle MFN2, SLC2A4 (formerly known as GLUT4), COX3 (encoding cytochrome c oxidase subunit III) and CS (encoding citrate synthase) mRNA levels were measured by real-time PCR. Results: Following biliopancreatic surgery, significant increases in MFN2 mRNA (from 0.4±0.2 to 1.7±1.1 arbitrary units [AU], p=0.019) and SLC2A4 mRNA (0.38±0.12 to 0.76±0.24 AU, p=0.04) were observed, while increases in COX3 mRNA (from 14.2±6.4 to 20.2±12.5 AU) and CS mRNA (from 0.4±0.1 to 0.7±0.3 AU) failed to reach statistical significance. Insulin-mediated whole-body glucose uptake significantly (p<0.0001) increased from 21.2±4.1 to 52.8±5.9 μmol kg fat-free mass −1 min −1 and glucose oxidation rose from 11.1±2.1 to 37.7±4.7 μmol kg fat-free mass −1 min −1 (p<0.0001). Levels of MFN2 mRNA were strongly correlated with the absolute values for the glucose oxidation rate, both during fasting (glucose oxidation =3.55 MFN2 mRNA + 3.93; R 2 =0.92, p<0.0001) and during the clamp (glucose oxidation=18.8 MFN2 mRNA+34.7; R 2 =0.80, p<0.0001). The percentage changes in MFN2 mRNA were positively correlated with the percentage change in glucose oxidation during the clamp (glucose oxidation percent (%) change=0.3 MFN2 mRNA percent (%) change +153.2; R 2 =0.61, p<0.001). Conclusions/interpretation: We propose that the significant increase in MFN2 mRNA levels may explain the increase in glucose oxidation observed in morbid obesity following bariatric surgery.

Sp1 is a key factor in maintaining basal Mfn2 transcription in VSMCs. Given the anti-proliferative actions of Mfn2, Sp1-induced Mfn2 transcription may represent a mechanism for prevention of VSMC proliferation and neointimal lesion and development.