Positive effect of evobrutinib in CNS remyelination models and lack of synergy with clemastine-A dose response study

Abstract

Background: To recover normal functions, remyelination in multiple sclerosis is crucial. Although endogenous remyelination occurs, it is often insufficient, and finding molecules promoting repair of demyelinated lesions is needed.

Objectives: To compare the remyelination potential of evobrutinib, an inhibitor of Bruton's tyrosine kinase and clemastine, an antagonist of M1 muscarinic acetylcholine receptor.

Methods: Remyelination was investigated in lysolecithin demyelinated organotypic mouse cerebellar slices and a transgenic Xenopus model of inducible-demyelination.

Results: Evobrutinib (100 nM) and clemastine (200 nM) potentiated remyelination of mouse cerebellar slices by a factor of 2.9 and 1.76, respectively. In conditionally demyelinated Xenopus, evobrutinib and clemastine increased remyelination by a factor of 1.61 and 1.92, respectively. Evobrutinib targets Bruton's tyrosine kinase expressed by microglia, and we showed that the increase in number of myeloid cells following demyelination is due to an extravasation from nearby vessels of macrophages migrating toward the optic nerve. In contrast, clemastine is expected to antagonize muscarinic receptor 1 expressing cells of the oligodendroglial lineage. We investigated a possible synergistic effect on remyelination by adding simultaneously both molecules. In both experimental models tested no significative improvement on remyelination of co-treatment with evobrutinib plus clemastine was observed.

Discussion: While evobrutinib increased 1.59 fold the number of microglia/macrophages, in the presence of clemastine the number of innate immune cells was decreased by 0.39 fold, therefore counteracting the beneficial effect of microglia/macrophages on remyelination.

Keywords: Myelin; demyelination; myelination; oligodendrocyte; remyelination.

Figure 1.

Spontaneous and drug-promoted remyelination of lysophosphatidylcholine (LPC) demyelinated mouse cerebellar slices. (A) Flow chart showing the sequence of events tested. Mouse cerebellar slices from Tg(plp:GFP) immunostained with a combination of anti-GFP (green) and anti-Calbindin (white) antibodies. After 6 DIV cerebellar slices are well myelinated (B). Demyelination was induced by LPC exposure for 15 h (C). Four days after removal of LPC, (11 DIV) slices are either spontaneously remyelinated (D) or following treatment with either clemastine (E) or evolbrutinib (F). Extent of remyelination by either clemastine (G) or evobrutinib (H) was evaluated by establishing the myelination index as described in the M&M section.

Figure 2.

Confocal imaging of Xenopus laevis Tg(mbp:GFP-ntr) optic nerve before demyelination, at the peak of demyelination and during recovery. (A) Flow chart showing the sequence of events tested. GFP reporter is expressed in myelinating oligodendrocytes before demyelination (B), at the peak of demyelination after 10 days of MTZ treatment (C) and during spontaneous recovery three days after the end of MTZ exposure (D) or treatment with either clemastine (E) or evobrutinib (F). Remyelination is evaluated by counting the number of oligodendrocytes (GFP+ cells) per optic nerve following either clemastine (G) or evobrutinib (H) treatment.

Figure 3.

Remyelination is not improved by co-treatment with clemastine and evobrutinib. (A) Spontaneous remyelination (CTRL) of lysophosphatidylcholine-demyelinated cerebellar slices was improved by treatment with either clemastine (200 nM) or evobrutinib (100 nM). Remyelination was not increased by simultaneous treatment with both molecules at the same concentration. No further improvement was observed by decreasing clemastine concentration. (B) In vivo, in MTZ-demyelinated Tg(mbp:GFP-ntr) optic nerve, remyelination was promoted by single administration of either clemastine (200 nM) or evobrutinib (100 nM), but no improvement by cotreatment with the two drugs and lowering clemastine concentration had no significative effect.

Figure 4.

Clemastine-inhibited microglia/macrophage. Tg(mbp:GFP-ntr) Xenopus laevis were exposed to MTZ and optic nerve stained with Alexa Fluor 647-conjugated isolectin IB4 (red microglia/macrophages) before being immunolabeled with anti-GFP (green oligodendrocyte). (A) before demyelination. (B, C) MTZ exposure is stopped after 10 days and animals are treated with either evobrutinib (B) or clemastine (C). (D) The number of IBA+ cells is increased by evobrutinib but dramatically decreased by clemastine.

Figure 5.

Live imaging of microglia/macrophages during demyelination. (A, A’) Brain tissue section of the transgenic Tg(mpeg1:mCherry) line (stage 50 tadpole) doubly stained for mCherry (A) and Alexa Fluor 488-conjugated isolectin IB4 (A’), showing the overlap of the two markers on microglia. (B) Movie No. 1 showing a mCherry+ microglial cells phagocyting GFP+ debris in the optic nerve of a stage 50 double transgenic Tg(Mbp:gfp-ntr)//(mpeg1:mCherry) undergoing demyelination due to a five days exposure to MTZ. Note the morphological change of microglia in comparison to A. (C, C’, C’’) GFP debris (white arrow) being phagocyted by a round shape mCherry microglia in the optic nerve of Tg(Mbp:gfp-ntr)//(mpeg1:mCherry). (D) extravasation of an mCherry+ macrophage (white arrow) from a blood vessel in the immediate vicinity of the NO. Successive images of an mCherry+ cell leaving a blood vessel and heading toward the optic nerve at different times T = 0s, T = 90s, T = 360s, T = 630s; the GFP+ oligodendrocyte is taken as a reference point (two perpendicular dotted white lines) which is immobile.