Bruton’s tyrosine kinase (BTK) inhibition promotes myelin repair in two different models of demyelination RESULTS Cellular expression of BTK and activated form BTK-phospho-Y223 (p-BTK) was determined by immunolabeling in normal adult mouse brain tissue sections and organotypic cerebellar slice cultures before and after lysophosphatidylcholine (LPC)-induced demyelination. and cerebellar slices was in sharp contras! with an 8.5-fold increase in the number of e cells observed in LPC demyelinated slice cultures. Double-labelling experiments confirmed that BTK-positive + cells were observed largely in lba1+ microglia i73.1%), as well as in a small fraction of s100 astrocytes F5,6%), with minimal + labelling in CC1 oligodendrocytes (< 1%). BTK was not detected in NeuN neurons or in Olig2 1cc1- oligodendrocyte precursor cells REFERENCES M.S. Aigrot 1 , E. Martin 1 , R. Grenningloh 2 , B. Stankoff 1,3 , C. Lubetzki 1,4 , U. Boschert 5 , B. Zalc 1 1 Sorbonne Université, Inserm, CNRS, Institut du Cerveau et de la Moelle Épinière, GH Pitié-Salpêtrière, Paris, France, 2 EMD Serono Research & Development Institute, Inc., Billerica, MA, United States, 3 AP-HP, Saint-Antoine Hospital, Paris, France, 4 AP-HP, Pitié-Salpêtrière Hospital, Paris, France, 5 Ares Trading S.A. an affiliate of Merck Serono S.A., Eysins, Switzerland Cont-plp:GFP-BTK LPC-plp:GFP-BTK We performed immunostaining for BTK (red) of cerebellar slices from P9 plp:GFP mice (green oligodendrocytes and myelin) before and after LPC treatment and observed an 8.5 fold increase in the number of cells with a detectable level of BTK. Similarly, p-BTK was also increased after LPC demyelination. (*p < 0.01; ***p < 0.0001). METHODS INTRODUCTION Remyelination was assayed at DIV13 after immunostaining with anti-PLP (ProteoLipid Protein myelin), anti-Caspr (paranodes) and anti-Calbindin (axons) antibodies. Note the increase in remyelination evaluated both by the number of PLP + internodes and the number of Caspr + paranodal structures in cerebellar slices from plp:GFP mice treated with an inhibitor of BTK (BTKi, MSC2494528) 1μM when compared to control conditions. (***p < 0.0001). Inhibition of BTK promotes remyelination ex vivo *** *** Caspr-PLP-Calb Caspr-PLP-Calb BTKi (MSC2494528) 1 μM RESULTS Inhibition of BTK promotes remyelination in vivo 6 DIV 9 DIV – 17h post LPC 13 DIV a c Cont LPC Iba1 BTK/Iba1 BTK/S100b p-BTK/CD11b BTK Cerebellar organotypic slices from P9 mice, were maintained in culture and submitted to LPC treatment at 6 days in vitro (DIV6) for 16-17 h. At DIV9 (i.e., peak of demyelination) immunostaining was performed for BTK (red), Iba1 (microglia, green). A) Small magnification of control (upper panel) and after demyelination (lower panel). B) Double-labelling experiments confirmed that BTK-positive cells were observed largely in Iba1 + microglia (73.1%), as well as in a small fraction of S100 + astrocytes (25.6%), with minimal labelling in CC1 + oligodendrocytes (<1%). BTK was not detected in NeuN + neurons nor in Olig2 + /CC1 - oligodendrocyte precursor cells. C) Higher magnification illustrating co-localisation of BTK and p-BTK in microglia and astrocytes A B C BTK BTK GFP GFP D0 D10 MBP GFP Nitroreductase We have developed a transgenic Xenopus laevis permitting live imaging of demyelination and remyelination by conditionally triggering ablation of myelin-forming oligodendrocytes. This line, MBP-GFP-NTR, expresses the green fluorescent protein (GFP) reporter fused to E. coli nitroreductase (NTR) under control of the mouse 1.9kb myelin basic protein (MBP) regulatory sequence. The NTR enzyme converts the nitro radical of prodrugs, such as metronidazole (MTZ), to a highly cytotoxic hydroxylamine derivative. In MTZ-exposed transgenic MBP-GFP-NTR tadpoles demyelination is restricted to the CNS, without axonal damage. At the end of MTZ exposure spontaneous remyelination occurs already after 3 days (R3). Under normal conditions BTK is expressed at a low level in microglial cells. After demyelination there is a clear increase in the level of expression of BTK, which is mostly expressed in microglial cells. BTK inhibition has a net effect on remyelination both ex vivo (rodent) and in vivo (xenopus). Our data suggest that BTK inhibition could represent a new therapeutic strategy to promote remyelination by targeting microglia. Ex vivo (myelinated organotypic cerebellar slice cultures) : In vivo (Xenopus laevis tadpoles) : A) Coronal sections from brain of stage 55 MBP-GFP-NTR tadpoles. Immunostaining for BTK (red), GFP (green) before (D0) and at the end of metronidazole treatment (D10). We observed an increase in the number of BTK + cells under demyelinated conditions (D10) compared to normal conditions (D0). B) Dose response of remyelination potency of BTKi (MSC2494528). Remyelination was assayed by counting the number of GFP+ cells per optic nerve in vivo on day 3 (R3) of the repair period. Of note BTKi (MSC2494528) treatment resulted in 1.7-fold improvement of remyelination compared to spontaneous recovery. (*p < 0.01; **p < 0.001). CONCLUSIONS Thetiot et al., J Vis Exp. 2019 Mannioui et al., Mult Scler. 2018. BTK/Iba1 A B Microglia are the resident macrophages of the Central nervous system (CNS). ln multiple sclerosis, microglia are the Janus of the innate immune response, exerting either a proinflammatory or a pro-regenerative function. Inhibition of Bruton’s tyrosine kinase (BTK), a member of the Tec family of kinases, blocks B-cell activation via the B cell receptor, myeloid activation via Fc receptors, and differentiation of proinflammatory macrophages in response to GM-CSF in vitro. However, the role of BTK in CNS, especially in CNS glia, is unknown. The aim of our study was to examine the cellular expression of BTK in the CNS and to investigate the consequences of BTK inhibition on remyelination both ex vivo and in vivo using two complementary experimental models of demyelination. Cont During ex vivo demyelination expression of BTK and p-BTK increases During ex vivo demyelination expression of BTK and p-BTK increases in microglial cells