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.

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.

Blocking mitochondrial calcium release in Schwann cells prevents demyelinating neuropathies.

Schwann cells produce myelin sheath around peripheral nerve axons. Myelination is critical for rapid propagation of action potentials, as illustrated by the large number of acquired and hereditary peripheral neuropathies, such as diabetic neuropathy or Charcot-Marie-Tooth diseases, that are commonly associated with a process of demyelination. However, the early molecular events that trigger the demyelination program in these diseases remain unknown. Here, we used virally delivered fluorescent probes and in vivo time-lapse imaging in a mouse model of demyelination to investigate the underlying mechanisms of the demyelination process. We demonstrated that mitochondrial calcium released by voltage-dependent anion channel 1 (VDAC1) after sciatic nerve injury triggers Schwann cell demyelination via ERK1/2, p38, JNK, and c-JUN activation. In diabetic mice, VDAC1 activity was altered, resulting in a mitochondrial calcium leak in Schwann cell cytoplasm, thereby priming the cell for demyelination. Moreover, reduction of mitochondrial calcium release, either by shRNA-mediated VDAC1 silencing or pharmacological inhibition, prevented demyelination, leading to nerve conduction and neuromuscular performance recovery in rodent models of diabetic neuropathy and Charcot-Marie-Tooth diseases. Therefore, this study identifies mitochondria as the early key factor in the molecular mechanism of peripheral demyelination and opens a potential opportunity for the treatment of demyelinating peripheral neuropathies.

In vivo time-lapse imaging of mitochondria in healthy and diseased peripheral myelin sheath.

The myelin sheath that covers a large amount of neurons is critical for their homeostasis, and myelinating glia mitochondria have recently been shown to be essential for neuron survival. However morphological and physiological properties of these organelles remain elusive. Here we report a method to analyze mitochondrial dynamics and morphology in myelinating Schwann cells of living mice using viral transduction and time-lapse multiphoton microscopy. We describe the distribution, shape, size and dynamics of mitochondria in live cells. We also report mitochondrial alterations in Opa1(delTTAG) mutant mice cells at presymptomatic stages, suggesting that mitochondrial defects in myelin contribute to OPA1 related neuropathy and represent a biomarker for the disease.

In vivo introduction of transgenes into mouse sciatic nerve cells in situ using viral vectors.

The myelin sheath is essential for the rapid and efficient propagation of action potentials. However, our understanding of the basic molecular mechanisms that regulate myelination, demyelination and remyelination is limited. Schwann cells produce myelin in the peripheral nervous system and remain associated with the axons of peripheral neurons throughout axonal migration to the target. Owing to the intimate relationship between these cell types it is difficult to fully reproduce their function in vitro. For this reason, we developed an approach based on the injection of an engineered virus into the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach can be used as an alternative to germline transgenesis to facilitate the investigation of peripheral nerve biology in vivo. The detailed protocol, described here, requires 3 weeks to complete. In comparison with genetic modification strategies, this protocol is a fast, reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo to investigate specific molecular mechanisms.

Identification of domains and amino acids essential to the collagen galactosyltransferase activity of GLT25D1.

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the ß1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166-168) and second motif (amino acids 461-463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein.

P120 catenin is required for thickening of Schwann cell myelin.

Schwann cell myelin is comprised of compacted membrane regions and cytoplasmic regions with non-compacted membrane. While adherens junctions (AJ) are abundant in non-compact regions, their role in the myelination process is largely undefined. To explore this issue, a small inhibitory hairpin RNA directed against p120ctn has been delivered using adenovirus infection of Schwann cells at early stages of myelination in vivo. With strong and specific reduction in p120ctn levels for over 2 months, (a) the adherens junctions of the infected cells were reduced in size and immature with respect to recruitment of alpha-catenin; and (b) the formation of Schmidt-Lanterman incisures was prevented and there was a marked reduction in the thickness of the myelin sheath without a change in internodal length. These data show that p120ctn is necessary in the myelinating Schwann cell for the formation of mature adherens junctions and a normal myelin sheath.

Adherens junctions in myelinating Schwann cells stabilize Schmidt-Lanterman incisures via recruitment of p120 catenin to E-cadherin.

Schwann cell myelin contains highly compacted layers of membrane as well as noncompacted regions with a visible cytoplasm. One of these cytoplasmic compartments is the Schmidt-Lanterman incisure, which spirals through the compacted layers and is believed to help sustain the growth and function of compact myelin. Incisures contain adherens junctions (AJs), the key components of which are E-cadherin, its cytoplasmic partners called catenins, and F-actin. To explore in vivo the role of cadherin and catenins in incisures, E-cadherin mutant proteins that completely replace endogenous cadherin have been delivered to the cells using adenovirus. When the introduced cadherin lacked its extracellular domain, association of p120 catenin (p120ctn) with the cadherin did not occur, and incisures disappeared. Remarkably, the additional replacement of two phosphorylatable tyrosines by phenylalanine in the cytoplasmic tail of the mutant cadherin restored both p120ctn binding and incisure architecture, indicating that p120ctn recruitment is critical for incisures maintenance and might be regulated by phosphorylations. In addition, the ability of the p120ctn/cadherin complex to support incisures was blocked by mutation of the Rho GTPase regulatory region of the p120ctn, and downregulation of Rac1 activity at the junction reversed this inhibition. Because Rho GTPases regulate the state of the actin filaments, these findings suggest that one role of p120ctn in incisures is to organize the cytoskeleton at the AJ. Finally, developmental studies of Schwann cells demonstrated that p120ctn recruitment from the cytoplasm to the AJ occurs before the appearance of Rac1 GTPase and F-actin at the junction.