- Molecular mechanisms of myelination/demyelination in peripheral nerves
Studying peripheral nerve myelination is not straightforward as the two cell types, myelinating cell and neuron, have to be intimate enough to build the tissue. We use two different models to analyze this phenomenon. First we co-culture purified Schwann cells and purified neurons together in order to obtain myelination in vitro. This approach, used also in other labs, allows quantifying the amount of myelinated segments produced by Schwann cells, and it permits to modify independently each cell type by transducing them with engineered viral vectors. The second approach we use allows to measure qualitative changes occurring during myelination in Schwann cells and in axons. In this approach, Schwann cells or neurons are transduced in the sciatic nerve of anesthetized animals with viral vectors. As myelination perturbations occur in an in vivo context fine morphological changes can be detected in light or electron microscopy.
An illustration of myelinated peripheral axons using GFP-expressing Schwann cells and drawed colored axons that cross them.
Using these approaches we investigated the role of adherens junctions in myelinating Schwann cells. We found that intact junctions are required to maintain the cell structure. In addition, we showed that a defect in the formation of these junctions during myelination induces a strong hypomyelination with a thin myelin sheath. This suggested that the organization of the cell structure is necessary to complete myelination. Adherens junctions are essential in the organization of polarized cells. The polarization of myelinating Schwann cells has been suggested some time ago but the nature and the function of this polarization had not been investigated in depth. We showed recently that the myelin sheath is polarized in a similar way as epithelial cells, with apical-like and basolateral-like domains.
We recently investigated also the role of conserved cell polarity genes during myelination and we found that Schwann cell myelin thickness and length is determined by the functional interplay of these genes. Moreover this polarization process is disturbed in cells of mice that model human Charcot-Marie-Tooth disease, suggesting that peripheral nerve diseases may result from a defect in Schwann cell polarization.
We are now pursuing our investigations on the role of cell polarity factors on myelination and their involvement in the etiology of peripheral nerve diseases. Our goal is to find key molecular pathways involved in human diseases etiology and find a way to modify them in order to propose therapies for these diseases.
- Roles of mitochondria in healthy and diseased myelin-ERC project
Peripheral neuropathies form a diverse group of pathologies that include diabetic and aging peripheral neuropathies and inherited Charcot-Marie-Tooth diseases. These highly debilitating diseases that affect peripheral nerves structure and function represent an increasing burden in European societies. While the etiologies of these diseases are diverse, defects in mitochondrial morphology and functions have recently emerged as one major cause for these diseases. Some mitochondrial dysfunctions are known to affect neurons, but most of them appear to affect the myelinating Schwann cell that produces the myelin sheath. Intriguingly the specific disruption of mitochondria in these glial cells does not directly affect myelination but induces neurodegeneration, suggesting that the role of glial mitochondria is complex. The goal of this project is to investigate the role of mitochondria in healthy and diseased myelin and to test whether we can change mitochondrial status and functions to prevent or treat these diseases.
Our working hypothesis is that glial mitochondria act as a homeostatic interface between axon and glia: they participate to the destabilization of Schwann cells during demyelination and they help to detoxify axons by scavenging reactive oxygen species produced by axonal mitochondria.
We have developed a novel approach that uses viral vectors to express cDNAs and/or small inhibitory RNAs in myelinating Schwann cells and myelinated axons in mice in vivo. I propose to use this approach combined with state-of-the-art imaging technique to challenge this preliminary concept in a meaningful in vivo context.
Viral tools will first be used to generate defects in particular mitochondrial functions in the myelinating Schwann cell. The impact on myelination and myelin maintenance will be assessed by light and electron microscopy. Second, viruses will be used to express genetically-encoded fluorescent probes designed to analyze mitochondrial status in living cells. This imaging approach will allow investigating mitochondrial status in healthy, demyelinating and diseased myelinating Schwann cells in vivo. Finally we will investigate the impact of glial mitochondria dysfunctions on the axon and on axonal mitochondria using fluorescent probes expressed in myelinated axons. Reversely we will also modify axonal mitochondria and check the impact of these changes on myelin and glial mitochondria.
As the impact of mitochondrial dysfunctions in diseases and aging of the human nervous system is attracting more attention, deciphering the role of mitochondria in myelinating cells is essential. Indeed the concept of homeostatic interface between axon and glia will be highly relevant to understand the molecular mechanisms of peripheral neuropathies but also of brain diseases such as multiple sclerosis, Alzheimer’s, Parkinson’s and Huntington’s diseases.
- Cell and gene therapy approaches for myelin diseases
Following our strong experience in viral vectors to transduce peripheral nerve cells, we are looking for novel viral vectors that could be used to modify myelinating and remyelinating Schwann cells in order to perform gene therapy attempt on animal models of Charcot-Marie-Tooth diseases.
Gonzalez S. et al., Nature Protocols, 9(5):1160-9, 2014
von Boxberg Y. et al., Glia, 2014 May 3
Bartolami S. et al., Med Sci (Paris), 28(4):341-3, 2012
Jacob C. et al., Nat. Neurosci, 14:429-436, 2011
Cotter L. et al., Science, 268:1415-18, 2010
Özçelik M. et al., J. Neurosci, 30(11): 4120-31, 2010
European patent registrated on March 21st, 2014 (N° EP14305402.1)
"Methods and Pharmaceutical Compositions for Enhancing Myelination"
Inventors: Nicolas Tricaud, Ruani Fernando, Laurent Cotter
- Anura Rambukkana, Université d’Edimbourg, Scotland
- Patrick Aubourg, INSERM U745/U986, Fontenay aux Roses, France
- Roman Chrast, Université de Lausanne, Suisse
- Fatiha Nothias, UPMC, Paris