BREaking the COde of MYelination – BRECOMY

Drosophila cells are of the nrv2Gal4UAScherry/APEX2 genotype. In this transgenic line, the two reporter genes mCherry and APEX2 are expressed by the sheathing glial cells (nrv2+). The Drosophila brain in larval stage III is dissected and the brain cells are dissociated. Depending on the developmental stage of the recipient, 5 to 16,000 cells are transplanted (about 500 to 1,600 glial cells) into the brain ventricle of tadpoles. The tadpoles come from the MBP-GFP-NTR transgenic line in which, thanks to the NTR transgene, we can induce an ablation of the myelinating oligodendrocytes and thus a conditional demyelination by introducing metronidazole into the swimming water (Kaya et al., J. Neurosci. 2012; Mannioui et al., Mult. Sclerosis 2018; Mannioui & Zalc, Methods Mol Biol. 2019). Thanks to the transparency of the Xenope tadpoles, the expression of mCherry, detected with a macroscope (AZ100 Nikon), makes it possible to follow, in vivo, the glial cells of Drosophila in the brain of Xenope. After fixation and treatment with DAB, these cells and their possible wrapping around axons are visualised under electron microscopy, after inclusion in the Epon and ultrafine sections (70nm, UC7 Leica Microsystem ultra microtome). The sections are mounted on 200mesh copper grids (Electron microscopy science cat# EMS200-Cu) then counter-stained with a lead citrate solution before being examined on the HT7700 electron microscope (Hitachi) at 70kV. The photos are taken on the integrated AMT XR41-B camera (2048X2048 pixels).

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The aim of these experiments is to establish whether Drosophila glial cells transplanted into the Xenope brain can i) survive and ii) myelinate Xenope axons. In the transgenic nrv2Gal4UAScherry/APEX2 line, the sheathing Drosophila glial cells express two reporter genes, mCherry and APEX2. The mCherry reporter allows Drosophila cells to be identified by fluorescence microscopy. The presence of mCherry cell bodies allows us to monitor the survival and migration of Drosophila cells in the Xenopic cerebral environment. We focus more particularly on elongated rectilinear structures suggestive of a myelin-like winding. The APEX2 reporter after reaction with DAB allows recognition of these same Drosophila structures under electron microscopy. Our first observations show images of relatively compact windings (up to 6 to 7 turns) around xenopic axons. Treatment of grafted tadpoles with metronidazole allows the removal of Xenopic oligodendrocytes (and myelin sheaths). The presence of DAB precipitate on the windings assures us of the Drosophila origin of these windings. These results, which are still preliminary, are encouraging, showing that Drosophila glial cells can not only survive and migrate in a Xenope environment, but that they can also form myelin «pseudo-sheaths« around Xenope axons. This suggests that the potential for myelination is carried by the axons.

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These results are preliminary and must at this stage remain confidential. They need to be confirmed and we will pay particular attention to studying the migration and survival of Drosophila cells transplanted into the brain of Xenopus. We will also study the importance and consequences of varying the transplant sites. In order to test factors that increase or decrease the ability of Drosophila sheathing cells to form pseudo-myelin sheaths around Xenope axons, we are developing co-culture systems of Xenope and Drosophila cells in parallel. On the one hand, we are developing conditions for myelinating Xenope cells and the possibilities of demyelinating these cultures, and on the other hand, we are looking for culture media favourable to both species. This work is in progress.

Rey S, Zalc B, Klämbt C. Evolution of glial wrapping: a new hypothesis [published online ahead of print, 2020 Mar 4]. Dev Neurobiol. 2020;10.1002/dneu.22739. doi:10.1002/dneu.22739
Lubetzki C, Zalc B, Williams A, Stadelmann C, Stankoff B. Remyelination in multiple sclerosis: from basic science to clinical translation. Lancet Neurol. (2020) in press.
Martin E, Aigrot MS, Grenningloh R, Stankoff B, Lubetzki C, Boschert U, Zalc B. Bruton’s tyrosine kinase inhibition promotes myelin repair. Brain Plasticity (2020) DOI 10.32.33/BPL-200100; in press.

It has been postulated that the emergence of vertebrates was made possible by the acquisition of neural crest cells, which then led to the development of evolutionarily advantageous complex head structures. In this regard, consecutive to the acquisition of the neural crest, the contribution of one important derivative—the myelin sheath—to the success of the vertebrates has to be pointed out. Without this structure, the vertebrates, as we know them, simply could not exist. Unfortunately, occurrence of demyelination in some diseases, the most frequent being Multiple Sclerosis (MS), can be deleterious and evolve into permanent handicap. Even though spontaneous remyelination, a crucial repair mechanism, occurs in MS, there is strong evidence that many MS lesions remain demyelinated despite abundance of oligodendroglial cells within or around the lesions. What prevents these cells from myelinating? Are axons lacking a necessary attractive signal or are the axons expressing some inhibitory molecules at their surface? Alternatively, is the oigodendroglial myelination program inhibited by some factors? Our ambition is to tackle this problem from a completely innovative angle. Myelin is present in the vast majority of vertebrates. In contrast, in insects, axons generally have only a single glial wrap and are not myelinated. Our working hypothesis is that breaking the code that triggers Drosophila ensheathing glia to form multiple wraps around axons will open the door to cues to repair MS lesions that otherwise do not remyelinate. Our research proposal will address the minimal requirements to induce a myelination program. The originality and innovation of our project relies on the uniqueness of the experimental model (associating non-myelinated invertebrate and myelinated vertebrate), a combination for which the two PIs (B. Zalc and C. Klämbt) have the unique competence and expertise.