Physiology of PNS axons relies on glycolytic metabolism in myelinating Schwann cells.

While lactate shuttle theory states that glial cells metabolize glucose into lactate to shuttle it to neurons, how glial cells support axonal metabolism and function remains unclear. Lactate production is a common occurrence following anaerobic glycolysis in muscles. However, several other cell types, including some stem cells, activated macrophages and tumor cells, can produce lactate in presence of oxygen and cellular respiration, using Pyruvate Kinase 2 (PKM2) to divert pyruvate to lactate dehydrogenase. We show here that PKM2 is also upregulated in myelinating Schwann cells (mSC) of mature mouse sciatic nerve versus postnatal immature nerve. Deletion of this isoform in PLP-expressing cells in mice leads to a deficit of lactate in mSC and in peripheral nerves. While the structure of myelin sheath was preserved, mutant mice developed a peripheral neuropathy. Peripheral nerve axons of mutant mice failed to maintain lactate homeostasis upon activity, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not altered but axonal mitochondria transport was slowed down, muscle axon terminals retracted and motor neurons displayed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment, which diverts pyruvate to mitochondrial oxidative phosphorylation, further aggravated motor dysfunction in mutant mice. Thus, lactate production through PKM2 enzyme and aerobic glycolysis is essential in mSC for the long-term maintenance of peripheral nerve axon physiology and function.

Mapping the N-Terminal Hexokinase-I Binding Site onto Voltage-Dependent Anion Channel-1 To Block Peripheral Nerve Demyelination.

The voltage-dependent anion channel (VDAC), the most abundant protein on the outer mitochondrial membrane, is implicated in ATP, ion and metabolite exchange with cell compartments. In particular, the VDAC participates in cytoplasmic and mitochondrial Ca2+ homeostasis. Notably, the Ca2+ efflux out of Schwann cell mitochondria is involved in peripheral nerve demyelination that underlies most peripheral neuropathies. Hexokinase (HK) isoforms I and II, the main ligands of the VDAC, possess a hydrophobic N-terminal structured in α-helix (NHKI) that is necessary for the binding to the VDAC. To gain further insight into the molecular basis of HK binding to the VDAC, we developed and optimized peptides based on the NHKI sequence. These modifications lead to an increase of the peptide hydrophobicity and helical content that enhanced their ability to prevent peripheral nerve demyelination. Our results provide new insights into the molecular basis of VDAC/HK interaction that could lead to the development of therapeutic compounds for demyelinating peripheral neuropathies.

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.

Dual imaging of dendritic spines and mitochondria in vivo reveals hotspots of plasticity and metabolic adaptation to stress.

Metabolic adaptation is a critical feature of synaptic plasticity. Indeed, synaptic plasticity requires the utilization and resupply of metabolites, in particular when the turnover is high and fast such as in stress conditions. What accounts for the localized energy burden of the post-synaptic compartment to the build up of chronic stress is currently not understood. We used in vivo microscopy of genetically encoded fluorescent probes to track changes of mitochondria, dendritic spines, ATP and H2O2 levels in pyramidal neurons of cortex before and after chronic unpredictable mild stress. Data revealed hotspots of postsynaptic mitochondria and dendritic spine turnover. Pharmacogenetic approach to force expression of the metabolic stress gene NR4A1 caused the fragmentation of postsynaptic mitochondria and loss of proximal dendritic spine clusters, whereas a dominant-negative mutant counteracted the effect of chronic stress. When fragmented, dendritic mitochondria produced lesser ATP at resting state and more on acute demand. This corresponded with significant production of mitochondrial H2O2 oxidative species in the dendritic compartment. Together, data indicate that pyramidal neurons adjust proximal dendritic spine turnover and mitochondria functions in keeping with synaptic demands.

AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A.

Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures.

LKB1 specifies neural crest cell fates through pyruvate-alanine cycling.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

In vivo real-time dynamics of ATP and ROS production in axonal mitochondria show decoupling in mouse models of peripheral neuropathies.

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.

Altered interplay between endoplasmic reticulum and mitochondria in Charcot-Marie-Tooth type 2A neuropathy.

Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot-Marie-Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum-mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.

The conceptual introduction of the "demyelinating Schwann cell" in peripheral demyelinating neuropathies.

Myelinating Schwann cells undergo irreversible demyelination in many demyelinating neuropathies that show complete demyelination of the internode. Dedifferentiation, reprogramming, and myelin clearance processes-which are specifically discussed in this article-appear to be shared by various demyelinating peripheral conditions, such as Wallerian degeneration, immune-mediated, and toxic demyelinating diseases. We propose to introduce the concept of the "demyelinating Schwann cell (DSC)" as a novel cell phenotype, which has specific properties required for myelin sheath clearance. We anticipate that the introduction of the DSC concept will provide a significant advance in understanding the pathophysiological mechanisms of demyelinating peripheral neuropathies.

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

Myelin sheath produced by Schwann cells covers and nurtures axons to speed up nerve conduction in peripheral nerves. Demyelinating peripheral neuropathies result from the loss of this myelin sheath and so far, no treatment exists to prevent Schwann cell demyelination. One major hurdle to design a therapy for demyelination is the lack of reliable measures to evaluate the outcome of the treatment on peripheral myelin in patients but also in living animal models. Non-linear microscopy techniques which include second harmonic generation (SHG), third harmonic generation (THG) and coherent anti-stokes Raman scattering (CARS) were used to image myelin ex vivo and in vivo in the sciatic nerve of healthy and demyelinating mice and rats. SHG did not label myelin and THG required too much light power to be compatible with live imaging. CARS is the most reliable of these techniques for in vivo imaging and it allows for the analysis and quantification of myelin defects in a rat model of CMT1A disease. This microscopic technique therefore constitutes a promising, reliable and robust readout tool in the development of new treatments for demyelinating peripheral neuropathies.

In Vivo Introduction of Transgenes into Mouse Sciatic Nerve Cells Using Viral Vectors.

Myelinated fibers are essential for the rapid and efficient propagation of nerve information throughout the body. These fibers result from an intimate crosstalk between myelinating glia and the myelinated axons and, because it is difficult to fully reproduce these interactions in vitro, the basic molecular mechanisms that regulate myelination, demyelination, and remyelination remain unclear. Schwann cells produce myelin in the peripheral nervous system (PNS) and remain associated with the axons of peripheral neurons throughout axonal migration to the target. In order to investigate more closely the biology of myelinated fibers, we developed a local transgenesis approach based on the injection of engineered viral vectors in the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach represents an alternative to germline modifications as it facilitates and speed up the investigation of peripheral nerve biology in vivo. Indeed the protocol we describe here requires just 3 weeks to complete. The injection of engineered viral vectors in the sciatic nerve of mice is a reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo in order to investigate specific molecular mechanisms.

A Combination of Ex vivo Diffusion MRI and Multiphoton to Study Microglia/Monocytes Alterations after Spinal Cord Injury.

Central nervous system (CNS) injury has been observed to lead to microglia activation and monocytes infiltration at the lesion site. Ex vivo diffusion magnetic resonance imaging (diffusion MRI or DWI) allows detailed examination of CNS tissues, and recent advances in clearing procedures allow detailed imaging of fluorescent-labeled cells at high resolution. No study has yet combined ex vivo diffusion MRI and clearing procedures to establish a possible link between microglia/monocytes response and diffusion coefficient in the context of spinal cord injury (SCI). We carried out ex vivo MRI of the spinal cord at different time-points after spinal cord transection followed by tetrahydrofuran based clearing and examined the density and morphology of microglia/monocytes using two-photon microscopy. Quantitative analysis revealed an early marked increase in microglial/monocytes density that is associated with an increase in the extension of the lesion measured using diffusion MRI. Morphological examination of microglia/monocytes somata at the lesion site revealed a significant increase in their surface area and volume as early as 72 hours post-injury. Time-course analysis showed differential microglial/monocytes response rostral and caudal to the lesion site. Microglia/monocytes showed a decrease in reactivity over time caudal to the lesion site, but an increase was observed rostrally. Direct comparison of microglia/monocytes morphology, obtained through multiphoton, and the longitudinal apparent diffusion coefficient (ADC), measured with diffusion MRI, highlighted that axonal integrity does not correlate with the density of microglia/monocytes or their somata morphology. We emphasize that differential microglial/monocytes reactivity rostral and caudal to the lesion site may thus coincide, at least partially, with reported temporal differences in debris clearance. Our study demonstrates that the combination of ex vivo diffusion MRI and two-photon microscopy may be used to follow structural tissue alteration. Lesion extension coincides with microglia/monocytes density; however, a direct relationship between ADC and microglia/monocytes density and morphology was not observed. We highlighted a differential rostro-caudal microglia/monocytes reactivity that may correspond to a temporal difference in debris clearance and axonal integrity. Thus, potential therapeutic strategies targeting microglia/monocytes after SCI may need to be adjusted not only with the time after injury but also relative to the location to the lesion site.

Wallerian demyelination: chronicle of a cellular cataclysm.

Wallerian demyelination is characteristic of peripheral nerve degeneration after traumatic injury. After axonal degeneration, the myelinated Schwann cell undergoes a stereotypical cellular program that results in the disintegration of the myelin sheath, a process termed demyelination. In this review, we chronologically describe this program starting from the late and visible features of myelin destruction and going backward to the initial molecular steps that trigger the nuclear reprogramming few hours after injury. Wallerian demyelination is a wonderful model for myelin degeneration occurring in the diverse forms of demyelinating peripheral neuropathies that plague human beings.

Myelinating Schwann Cell Polarity and Mechanically-Driven Myelin Sheath Elongation.

Myelin sheath geometry, encompassing myelin sheath thickness relative to internodal length, is critical to optimize nerve conduction velocity and these parameters are carefully adjusted by the myelinating cells in mammals. In the central nervous system these adjustments could regulate neuronal activities while in the peripheral nervous system they lead to the optimization and the reliability of the nerve conduction velocity. However, the physiological and cellular mechanisms that underlie myelin sheath geometry regulation are not yet fully elucidated. In peripheral nerves the myelinating Schwann cell uses several molecular mechanisms to reach and maintain the correct myelin sheath geometry, such that myelin sheath thickness and internodal length are regulated independently. One of these mechanisms is the epithelial-like cell polarization process that occurs during the early phases of the myelin biogenesis. Epithelial cell polarization factors are known to control cell size and morphology in invertebrates and mammals making these processes critical in the organogenesis. Correlative data indicate that internodal length is regulated by postnatal body growth that elongates peripheral nerves in mammals. In addition, the mechanical stretching of peripheral nerves in adult animals shows that myelin sheath length can be increased by mechanical cues. Recent results describe the important role of YAP/TAZ co-transcription factors during Schwann cell myelination and their functions have linked to the mechanotransduction through the HIPPO pathway and the epithelial polarity factor Crb3. In this review the molecular mechanisms that govern mechanically-driven myelin sheath elongation and how a Schwann cell can modulate internodal myelin sheath length, independent of internodal thickness, will be discussed regarding these recent data. In addition, the potential relevance of these mechanosensitive mechanisms in peripheral pathologies will be highlighted.

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

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

Fast 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.

Assembly of juxtaparanodes in myelinating DRG culture: Differential clustering of the Kv1/Caspr2 complex and scaffolding protein 4.1B.

The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination.