Traumatic and Diabetic Schwann Cell Demyelination Is Triggered by a Transient Mitochondrial Calcium Release through Voltage Dependent Anion Channel 1

A large number of peripheral neuropathies, among which are traumatic and diabetic peripheral neuropathies, result from the degeneration of the myelin sheath, a process called demyelination. Demyelination does not result from Schwann cell death but from Schwann cell dedifferentiation, which includes reprograming and several catabolic and anabolic events. Starting around 4 h after nerve injury, activation of MAPK/cJun pathways is the earliest characterized step of this dedifferentiation program. Here we show, using real-time in vivo imaging, that Schwann cell mitochondrial pH, motility and calcium content are altered as soon as one hour after nerve injury. Mitochondrial calcium release occurred through the VDAC outer membrane channel and mPTP inner membrane channel. This calcium influx in the cytoplasm induced Schwann-cell demyelination via MAPK/c-Jun activation. Blocking calcium release through VDAC silencing or VDAC inhibitor TRO19622 prevented demyelination. We found that the kinetics of mitochondrial calcium release upon nerve injury were altered in the Schwann cells of diabetic mice suggesting a permanent leak of mitochondrial calcium in the cytoplasm. TRO19622 treatment alleviated peripheral nerve defects and motor deficit in diabetic mice. Together, these data indicate that mitochondrial calcium homeostasis is instrumental in the Schwann cell demyelination program and that blocking VDAC constitutes a molecular basis for developing anti-demyelinating drugs for diabetic peripheral neuropathy

Label-free non-linear microscopy to measure myelin outcome in a rodent model of Charcot-Marie-Tooth diseases

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Optimal myelin elongation relies on YAP activation by axonal growth and inhibition by Crb3/Hippo pathway

International audienceFast nerve conduction relies on successive myelin segments that electrically isolate axons. Segment geometry-diameter and length-is critical for the optimization of nerve conduction and the molecular mechanisms allowing this optimized geometry are partially known. We show here that peripheral myelin elongation is dynamically regulated by stimulation of YAP (Yes-associated protein) transcription cofactor activity during axonal elongation and limited by inhibition of YAP activity via the Hippo pathway. YAP promotes myelin and non-myelin genes transcription while the polarity protein Crb3, localized at the tips of the myelin sheath, activates the Hippo pathway to temper YAP activity, therefore allowing for optimal myelin growth. Dystrophic Dy(2j/2j) mice mimicking human peripheral neuropathy with reduced internodal lengths have decreased nuclear YAP which, when corrected, leads to longer internodes. These data show a novel mechanism controlling myelin growth and nerve conduction, and provide a molecular ground for disease with short myelin segments

Mechanical stimulation and mechanobiological characterization of cartilage micropellets with a single and new custom-made device

National audienceBackground. Articular cartilage is a tissue with poor self-repair capacity, which is prone to progressive destruction after injuries. In cartilage, the chondrocytes surrounded by their extracellular matrix are organized in a complex structure that plays an essential weight-bearing role in the joint and can both sense and respond to various mechanical stimuli. Cartilage micropellet is a relevant and widely used in vitro model to study cartilage growth but poorly investigated in terms of mechanical characterization because of its small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of mesenchymal stromal cells (MSCs)-derived cartilage micropellets. Methods. Alginate beads were made with a solution of 3 % sodium alginate extruded into 0,1 M CaCl2. Collagen microspheres were prepared with type I collagen in hydrofluoroether. Human bone marrow-derived MSCs were differentiated into chondrocytes by culture in micropellets with 10 ng/mL TGFβ3- containing inductive medium for 21 days. The fluidic-based device was designed for the concomitant culture of six micropellets placed into the conical wells of a chamber where they were stimulated by a positive pressure (sinusoidal, square or constant). The sinking of each micropellet into the cone and its deformation were recorded by a camera. Expression of the chondrocyte markers SOX9, AGG and COL2B were quantified 24 hours after stimulation by RT-qPCR. The Young’s modulus was determined using a finite element model employing a neo-Hookean hyperelastic law. Results. Alginate- and collagen-based microspheres were first used to validate the reliability of the device. Repeatability and reproducibility of pressure signals used for mechanical stimulation were demonstrated. The mechanical properties of the microspheres were equivalent to those determined by a conventional compression test and shown to be reproducible. MSC-derived cartilage micropellets were stimulated with sinusoidal, square or constant pressure. Different parameters (amplitude, frequency, duration) of the square pressure signal were tested on the expression of chondrocyte markers. A stimulation of 1 Hz at 7 kPa for 30 min induced a significant increase of chondrocyte markers. The mechanical properties of the micropellets were measured and a Young’s modulus of 47.6 ± 26.2 kPa was determined. Conclusion. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with radius in range of 600 to 1300 μm. Of importance in case of MSC-based cartilage micropellets, mechanical stimulation can be performed in parallel on six microspheres allowing the molecular and mechanical characterization on the same group of samples. In the future, the device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli in a longitudinal study

Mechanical stimulation and mechanobiological characterization of cartilage micropellets with a single and new custom-made device

National audienceBackground. Articular cartilage is a tissue with poor self-repair capacity, which is prone to progressive destruction after injuries. In cartilage, the chondrocytes surrounded by their extracellular matrix are organized in a complex structure that plays an essential weight-bearing role in the joint and can both sense and respond to various mechanical stimuli. Cartilage micropellet is a relevant and widely used in vitro model to study cartilage growth but poorly investigated in terms of mechanical characterization because of its small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of mesenchymal stromal cells (MSCs)-derived cartilage micropellets. Methods. Alginate beads were made with a solution of 3 % sodium alginate extruded into 0,1 M CaCl2. Collagen microspheres were prepared with type I collagen in hydrofluoroether. Human bone marrow-derived MSCs were differentiated into chondrocytes by culture in micropellets with 10 ng/mL TGFβ3- containing inductive medium for 21 days. The fluidic-based device was designed for the concomitant culture of six micropellets placed into the conical wells of a chamber where they were stimulated by a positive pressure (sinusoidal, square or constant). The sinking of each micropellet into the cone and its deformation were recorded by a camera. Expression of the chondrocyte markers SOX9, AGG and COL2B were quantified 24 hours after stimulation by RT-qPCR. The Young’s modulus was determined using a finite element model employing a neo-Hookean hyperelastic law. Results. Alginate- and collagen-based microspheres were first used to validate the reliability of the device. Repeatability and reproducibility of pressure signals used for mechanical stimulation were demonstrated. The mechanical properties of the microspheres were equivalent to those determined by a conventional compression test and shown to be reproducible. MSC-derived cartilage micropellets were stimulated with sinusoidal, square or constant pressure. Different parameters (amplitude, frequency, duration) of the square pressure signal were tested on the expression of chondrocyte markers. A stimulation of 1 Hz at 7 kPa for 30 min induced a significant increase of chondrocyte markers. The mechanical properties of the micropellets were measured and a Young’s modulus of 47.6 ± 26.2 kPa was determined. Conclusion. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with radius in range of 600 to 1300 μm. Of importance in case of MSC-based cartilage micropellets, mechanical stimulation can be performed in parallel on six microspheres allowing the molecular and mechanical characterization on the same group of samples. In the future, the device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli in a longitudinal study

Degradable double hydrophilic block copolymers and tripartite polyionic complex micelles thereof for small interfering ribonucleic acids (siRNA) delivery

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Chitosan-PNIPAM Thermogel Associated with Hydrogel Microspheres as a Smart Formulation for MSC Injection

Regenerative medicine based on cell therapy has emerged as a promising approach for the treatment of various medical conditions. However, the success of cell therapy heavily relies on the development of suitable injectable hydrogels that can encapsulate cells and provide a conducive environment for their survival, proliferation, and tissue regeneration. Herein, we address the medical need for cyto- and biocompatible injectable hydrogels by reporting on the synthesis of a hydrogel-forming thermosensitive copolymer. The copolymer was synthesized by grafting poly(N-isopropylacrylamide-co-carboxymethyl acrylate) (PNIPAM-COOH) onto chitosan through amide coupling. This chemical modification resulted in the formation of hydrogels that exhibit a sol–gel transition with an onset at approximately 27 °C, making them ideal for use in injectable applications. The hydrogels supported the survival and proliferation of cells for several days, which is critical for cell encapsulation. Furthermore, the study evaluates the addition of collagen/chitosan hybrid microspheres to support the adhesion of mesenchymal stem cells within the hydrogels. Altogether, these results demonstrate the potential of the PNIPAM-chitosan thermogel for cell encapsulation and its possible applications in regenerative medicine