Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy

: SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired

Microelectrochemical patterning of gold surfaces using 4-azidobenzenediazonium and scanning electrochemical microscopy

This work describes for the first time the possibility of performing local micro electrochemical grafting of a gold substrate by 4-azidobenzenediazonium by SECM in a single and simple one step without complications from adsorption. The electrografted spots of diazonium were performed by positioning a Pt tip at a given distance above the gold substrate and the SECM was used in a three-electrode configuration (the Pt tip serving as the microanode) in acetonitrile containing 5 mM 4-azidobenzenediazonium and 0.1 M Bu4NBF4 during 10 ms. The dimensions of the derivatized areas of the substrates were finely tuned by using different experimental conditions (tip distance above the substrate, tip diameter, presence or absence of supporting electrolyte). The use of the azido-derivated diazonium molecule and these preliminary results open the gate to important applications and developments devoted to the local micro functionalization of electrodes by thin layers that allow the implementation of the emerging and attractive interfacial click reaction. Keywords: Diazonium, Electrografting, Spots, Electrochemical scanning microscopy, Local derivatizatio

Microelectrochemical patterning of gold surfaces using 4-azidobenzenediazonium and scanning electrochemical microscopy

This work describes for the first time the possibility of performing local micro electrochemical grafting of a gold substrate by 4-azidobenzenediazonium by SECM in a single and simple one step without complications from adsorption. The electrografted spots of diazonium were performed by positioning a Pt tip at a given distance above the gold substrate and the SECM was used in a three-electrode configuration (the Pt tip serving as the microanode) in acetonitrile containing 5 mM 4-azidobenzenediazonium and 0.1 M Bu4NBF4 during 10 ms. The dimensions of the derivatized areas of the substrates were finely tuned by using different experimental conditions (tip distance above the substrate, tip diameter, presence or absence of supporting electrolyte). The use of the azido-derivated diazonium molecule and these preliminary results open the gate to important applications and developments devoted to the local micro functionalization of electrodes by thin layers that allow the implementation of the emerging and attractive interfacial click reaction

Flexible, Biocompatible PET Sheets: A Platform for Attachment, Proliferation and Differentiation of Eukaryotic Cells

Transparent, flexible, biaxially oriented polyethylene terephthalate (PET) sheets were modified by bioactive polymer-fibronectin top layers for the attachment of cells and growth of muscle fibers. Towards this end, PET sheets were grafted with 4-(dimethylamino)phenyl (DMA) groups from the in situ generated diazonium cation precursor. The arylated sheets served as macro-hydrogen donors for benzophenone and the growth of poly(2-hydroxy ethyl methacrylate) (PHEMA) top layer by surface-confined free radical photopolymerization. The PET-PHEMA sheets were further grafted with fibronectin (FBN) through the 1,1-carbonyldiimidazole coupling procedure. The bioactive PET-PHEMA-I-FBN was then employed as a platform for the attachment, proliferation and differentiation of eukaryotic cells which after a few days gave remarkable muscle fibers, of ~120 µm length and ~45 µm thickness. We demonstrate that PET-PHEMA yields a fast growth of cells followed by muscle fibers of excellent levels of differentiation compared to pristine PET or standard microscope glass slides. The positive effect is exacerbated by crosslinking PHEMA chains with ethylene glycol dimethacrylate at initial HEMA/EGDA concentration ratio = 9/1. This works conclusively shows that in situ generated diazonium salts provide aryl layers for the efficient UV-induced grafting of biocompatible coating that beneficially serve as platform for cell attachment and growth of muscle fibers

Flexible, Biocompatible PET Sheets: A Platform for Attachment, Proliferation and Differentiation of Eukaryotic Cells

Transparent, flexible, biaxially oriented polyethylene terephthalate (PET) sheets were modified by bioactive polymer-fibronectin top layers for the attachment of cells and growth of muscle fibers. Towards this end, PET sheets were grafted with 4-(dimethylamino)phenyl (DMA) groups from the in situ generated diazonium cation precursor. The arylated sheets served as macro-hydrogen donors for benzophenone and the growth of poly(2-hydroxy ethyl methacrylate) (PHEMA) top layer by surface-confined free radical photopolymerization. The PET-PHEMA sheets were further grafted with fibronectin (FBN) through the 1,1-carbonyldiimidazole coupling procedure. The bioactive PET-PHEMA-I-FBN was then employed as a platform for the attachment, proliferation and differentiation of eukaryotic cells which after a few days gave remarkable muscle fibers, of ~120 µm length and ~45 µm thickness. We demonstrate that PET-PHEMA yields a fast growth of cells followed by muscle fibers of excellent levels of differentiation compared to pristine PET or standard microscope glass slides. The positive effect is exacerbated by crosslinking PHEMA chains with ethylene glycol dimethacrylate at initial HEMA/EGDA concentration ratio = 9/1. This works conclusively shows that in situ generated diazonium salts provide aryl layers for the efficient UV-induced grafting of biocompatible coating that beneficially serve as platform for cell attachment and growth of muscle fibers

Organic Layers Bonded to Industrial, Coinage, and Noble Metals through Electrochemical Reduction of Aryldiazonium Salts

The reduction of diazonium salts in an aprotic medium permits the attachment of substituted aryl groups to a variety of metals:  Co, Ni, Cu, Zn, Pt, and Au. These aryl groups are strongly bonded to the metal as they resist sustained rinsing under sonication in organic solvents. The organic layers have been characterized by cyclic voltammetry, infrared reflection absorption spectroscopy, X-ray photoelectron spectroscopy, Rutherford backscattering, electrochemical impedance spectroscopy, and atomic force microscopy. From these data it is possible to propose a structure for these grafted layers

Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy

<div><p>Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the <i>desmin</i> gene (<i>DES</i>), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant <i>DES</i>. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.</p></div

Coating conductive or semiconductive materials by polymerization of monomers on the surfaces.

Conductive and semiconductive materials are coated with 0.1-100-μm polymer anticorrosive films by (1) grafting diazonium salts having groups that function as polymn. initiators onto the surface and (2) radical or ring-opening polymn. of the monomers on the surface. Thus, an Fe electrode was electrolytically grafted with p-BrCHMeC6H4N2BF4, and Me methacrylate was polymd. by ATRP on the grafted surface

Stimulation of autophagy with PP242 reduces desmin aggregation in myoblasts.

<p>(A) C2C12 cells were transiently transfected for 4 h with GFP-Desmin D399Y mutant, washed, and treated for 16 h with PP242 (10 μM). They were fixed and several fields photographed. A typical field is displayed. Green dots are cell aggregates and blue dots are cell nuclei visualized with DAPI. Cells from the control panel (CNTL) were treated with DMSO. Scale bar, 30 μm. (B) Quantification of 3 independent experiments (n = 1200 total cells for each condition in each experiment). The left panel displays results obtained with the GFP-Desmin Q389P construct, and in the right panel, from GFP-Desmin D399Y transfections. (C) Same as for (B), except that myc-Desmin Q389P (left panel) and myc-Desmin D399Y (right panel) constructs were used. Cells were transiently transfected for 4 h with these constructs, washed, and treated 16 h with PP242 (10 μM) or DMSO as solvent (CNTL). They were fixed and then processed for immunocytochemistry with an anti-myc antibody. Numbers of cells with aggregates per field and number of myc-positive cells without aggregates but displaying a normal desmin network were counted. The percentage of cells with aggregates among all myc-positive cells was calculated, taking into account 5 fields per experimental condition (n = 1400) in 3 independent experiments. Transfection efficiency was 25%. Significant differences from the control are indicated with asterisk (p<0.05 calculated with a non-parametric test).</p