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Mitochondria are critical for the function and maintenance of myelinated axons notably through Adenosine triphosphate (ATP) production. A direct by-product of this ATP production is reactive oxygen species (ROS), which are highly deleterious for neurons. While ATP shortage and ROS levels increase are involved in several neurodegenerative diseases, it is still unclear whether the real-time dynamics of both ATP and ROS production in axonal mitochondria are altered by axonal or demyelinating neuropathies. To answer this question, we imaged and quantified mitochondrial ATP and hydrogen peroxide (H2O2) in resting or stimulated peripheral nerve myelinated axons in vivo, using genetically-encoded fluorescent probes, two-photon time-lapse and CARS imaging. We found that ATP and H2O2 productions are intrinsically higher in nodes of Ranvier even in resting conditions. Axonal firing increased both ATP and H2O2 productions but with different dynamics: ROS production peaked shortly and transiently after the stimulation while ATP production increased gradually for a longer period of time. In neuropathic MFN2R94Q mice, mimicking Charcot-Marie-Tooth 2A disease, defective mitochondria failed to upregulate ATP production following axonal activity. However, elevated H2O2 production was largely sustained. Finally, inducing demyelination with lysophosphatidylcholine resulted in a reduced level of ATP while H2O2 level soared. Taken together, our results suggest that ATP and ROS productions are decoupled under neuropathic conditions, which may compromise axonal function and integrity. Electronic supplementary material The online version of this article (10.1186/s40478-019-0740-4) contains supplementary material, which is available to authorized users.

National audience; Background. 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.

Summary Whether glial cells use a particular metabolism to support axonal metabolism and function remains controversial. We show here that the deletion of PKM2, an enzyme essential for the Warburg effect, in mature myelinating Schwann cells (mSC) of mice leads to a deficit of lactate in these cells and in peripheral nerves, and to motor defects despite no alteration of the myelin sheath. When electrically stimulated, peripheral nerve axons of mutant mice failed to maintain lactate homeostasis, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not changed but axonal mitochondria transport was altered, muscle axon terminals retracted and motor neurons showed cellular stress. Further reducing lactate availability through dichloroacetate treatment definitely aggravated axonal malfunction in mutant mice. Thus, cancer-like Warburg effect is essential in mSC for the long term maintenance of peripheral nerve axons physiology and function. One Sentence Summary Lactate-dependent axons rely on cancer-like metabolism in myelinating Schwann cells.

Impact of extramitochondrial ROS on mito-roGFP-Orp1 during demyelination. Upon injection of CellROX deep red into the sciatic nerve, fluorescence signal could be detected indicative of oxidative stress. No significant differences were observed between the time points of the demyelination process, nor nerves injected with PBS. In the contralateral nerves, which were not injected with LPC or PBS, a fluorescence signal was detected as well. Before CellROX injection, no fluorescence signal was observed. At the injection site, where tissue is locally damaged due to insertion of the syringe, a much stronger fluorescence signal is observed than at the region where mitochondrial H2O2 was measured (Fig. 6c). (PDF 4122 kb)

LPC injection induces degeneration of the myelin sheath, but not axonal degeneration. In a healthy sciatic nerve (Before injection), axons (red) are surrounded by a myelin sheath (green). This myelin sheath is severely damaged following injection of LPC into the sciatic nerve (LPC + 1w), but no sign of axonal degeneration. Injection of PBS (PBS + 1w) does not affect axon or myelin sheath physiology. Scale = 10 μm. (PDF 4147 kb)

LPC injection induces degeneration of the myelin sheath, but not axonal degeneration. In a healthy sciatic nerve (Before injection), axons (red) are surrounded by a myelin sheath (green). This myelin sheath is severely damaged following injection of LPC into the sciatic nerve (LPC + 1w), but no sign of axonal degeneration. Injection of PBS (PBS + 1w) does not affect axon or myelin sheath physiology. Scale = 10 μm. (PDF 4147 kb)