Bruton’s tyrosine kinase inhibition promotes myelin repair

E. Martin*1, M.S. Aigrot*1, R. Grenningloh2, B. Stankoff1,3, C. Lubetzki1,4, U. Boschert5, B. Zalc1.

1Sorbonne Université, Inserm, CNRS, Institut du Cerveau et de la Moelle Épinière, GH Pitié-Salpêtrière, F-75013 Paris, France ; 2EMD Serono Research & Development Institute, Inc., Billerica, MA, United States ;3AP-HP, Saint-Antoine Hospital, F-75012 Paris, France ;4AP-HP, GH Pitié-

Salpêtrière, F-75013 Paris, France ; 5Ares Trading S.A. an affiliate of Merck Serono S.A., Eysins, Switzerland.*These authors contributed equally to the work

INTRODUCTION

Microglia are the resident macrophages of the central nervous system (CNS). In multiple sclerosis (MS) and relatedexperimental models, microglia have either a proinflammatory or a proregenerative/pro-remyelinating function.Inhibition of Bruton’s tyrosine kinase (BTK), a member of the Tec family of kinases, has been shown to blockdifferentiation of proinflammatory macrophages in response to granulocyte–macrophage colony-stimulating factor invitro. However, the role of BTK in the CNS is unknown. Our first aim was to investigate the cellular pattern ofexpression of BTK in the mouse CNS, which was unknown. Then we investigated the effect of BTK inhibitors onremyelination by taking advantage of an ex-vivo model of mouse cerebellar slices, allowing to monitor theremyelination process after lysolecithin-induced demyelination. The third aim was to complement our ex vivoinvestigations, by using an in vivo conditional demyelination model developed in Xenopus laevis : the transgenic MBP-GFP-NTR line, which has been shown to be well adapted to screen pro-remyelinating compounds

METHODS

9 DIV – 17h post LPC 13 DIV

MBP GFP Nitroreductase

6 DIV

1°) Mouse cerebellar slice preparation : Sagittal slices of cerebellum (200–300 μm thick) were cut using tissue chopper and placed on organotypic culture inserts. After 6 days inculture, demyelination was induced by addition of 0.5 mg/ml lysophosphatidylcholine (LPC) to the medium for 16–17 h. Then slices were transferred back to normal medium. At DIV9 (thepeak of demyelination), BTK inhibitors was added for 2 days and renewed at DIV11. Quantification of spontaneous remyelination (control) was compared to remyelination in the presenceof BTK inhibitors at DIV13. 2°) pMBP-GFP-NTR transgenic Xenopus laevis : MBP-GFP-NTR line, 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 highlycytotoxic hydroxylamine derivative. In MTZ-exposed transgenic MBP-GFP-NTR tadpoles demyelination is restricted to the CNS, without axonal damage. At D10, at the end of MTZ exposureBTK inhibitors was added for 3 days and renewed every day. Quantification of spontaneous remyelination (control) was compared to remyelination in the presence of BTK inhibitors at R3.

1°) Ex vivo (myelinated organotypic cerebellar slice cultures) :

2°) In vivo (Xenopus laevis tadpoles) :

RESULTS

BTK and p-BTK expression increased after demyelination

Increase BTK signal upon demyelination : Cerebellar organotypic slices from P9 Plp-GFP mouse (green oligodendrocytes and myelin), were maintained in culture (A) or submitted to LPCtreatment at 6 days in vitro (DIV) for 16-17 h (B) before being immuno-stained for BTK (red) or p-BTK at 9DIV. LPC treatment induced a demyelination (loss of GFP signal B, C) and aconcomitant 8.5-fold increase in BTK signal (B, D). Similarly, p-BTK was also increased after LPC demyelination (E). (*p < 0.01; ***p < 0.0001). Scale bar: 100µm

RESULTS

Cellular expression of BTK and p-BTK

BTK Iba1 Merge

BTK Iba1 Merge

BTK was detected in microglia and astrocyte : Cerebellar organotypic slices from P9 mouse, were maintained in culture and submitted to LPC treatment at 6 DIV for 16-17 h. At DIV9 (i.e.,peak of demyelination) immunostaining was performed for BTK (Red) and Iba1 (microglia, green). A-F) Small magnification of control (A-C), and after LPC-induced demyelination (D-F). G,H,J,K)Higher magnification illustrating co-localisation of BTK and p-BTK in microglia labeled with Iba1 (G) or CD11-F4/80-CD68 (J) and astrocyte S100ß+ (H, K). L) An example of two cells co-expressing BTK and p-BTK. Note that BTK is more at the surface while p-BTK is more intracytoplasmic. I, M) Quantification of different cells co-expressing BTK (I) or p-BTK (M). Scale bar: A-F =20µm; G, H, J, K, L=10µm

Remyelination in cerebellar organotypic slices after BTKi treatment.

A BEHAVIORAL TEST TO EVALUATE THE FUNCTIONAL CONSEQUENCES OF DEMYELINATION IN A CONDITIONAL XENOPUS LAEVIS MODEL.

RESULTS

INTRODUCTION

Our transgenic Xenopus model MBP-GFP-NTR enables us to both label mature oligodendrocytes and induce conditional demyelination. Demyelination and spontaneous remyelination are studied in vivo in the optic nerve thanks to the transparency of tadpoles.

What is the functional impact of demyelination and is there a

functional recovery following remyelination ? We reasoned that demyelination should translate into loss of sensory-motor functions. Since

the optic nerve is myelinated we have adapted a visual avoidance paradigm in MBP-GFP-NTR Xenopus tadpoles to test the behavioral consequences of demyelination.

METHODS

CONCLUSIONS

• Demyelination (evaluated by loss of oligodendrocytes) results in a

sensory-motor deficit : swimming is altered and tadpoles are less reactive to virtual collisions.

• Spontaneous myelin repair leads to apparent functional recovery. • A larger number of animals needs to be analyzed to reach statistical

significance.

REFERENCES

Kaya F, et al. "Live imaging of targeted cell ablation in Xenopus: a new model to study demyelination and repair. " J Neurosci (2012) 32(37):12885-95

Khakhalin A, et al. "Excitation and inhibition in recurrent networks mediate collision avoidance

in Xenopus tadpoles." European Journal of Neuroscience (2014) 40(6):2948-62.

Dong W, et al. "Visual avoidance in Xenopus tadpoles is correlated with the maturation of visual responses in the optic tectum." Journal of neurophysiology (2009) 101(2):803-15.

Transgenic Xenopus MBP-GFP-NTR tadpoles stages NF52-53 are treated with metronidazole during 10 days and then

returned in standard rearing medium.

Virtual collision test setup

Timelapse of escape response

Successful avoidance was determined as an acceleration >50cm/s2 in proximity to

the black dot (1-1.3cm) corroborated by a change in direction.

TIMEPOINTS :

• Behavioral tests evaluating

swimming behavior during 30s

and visual avoidance to virtual collisions (6 tryouts) AM before

feeding

• Counting of GFP+ cells in

each optic nerve PM

MBP immunostaining (red) of coronal tissue sections of brainstem

Optic nerve confocal microscopy

Swimming behavior excluding escape responses during 30s

SWIMMING BEHAVIOR IS ALTERED FOLLOWING DEMYELINATION

VISUAL AVOIDANCE PERFORMANCE DECLINES AFTER DEMYELINATION

Esther Henriet1, Abdelkrim Mannioui1, Nadège Krebs2, Arseny Khakhalin3 and Bernard Zalc1 1Team « Oligodendrocyte development and neurovascular interactions » , Sorbonne Universités UPMC Univ Paris 06, Inserm, CNRS, ICM-GH Pitié-Salpêtrière, 75013 Paris, France; 2Noldus IT, Wageningen, The Netherlands; 3Biology Program, Bard College, Annandale-on-Hudson, NY, USA

t : 0.00s t : 0.03s

t : 0.06s t : 0.09s

t : 0.12s t : 0.15s

n=11

n=14

n=23

n=29

n=23

n=44

n=23

n=44

n=8

n=8

n=8

n=4

D10

D0

R3

METRONIDAZOLE-INDUCED CONDITIONAL DEMYELINATION IS FOLLOWED BY SPONTANEOUS REMYELINATION

Inhibition of BTK favors remyelination : A, B) Cerebellar organotypic slices from P9 mouse, were exposed to LPC at 6 DIV for 16-17 h and treated with BTKi (MSC2494528) (1µM) fromDIV 9 before triple labeling with anti-PLP (myelinated internodes, green), anti-Caspr (paranodes, white) and anti-Calbindin (axons, red) antibodies. Small white arrows in control (A) and BTKitreated (B) cultures point to some complete nodal structures with the Caspr+ paranode on each side of the Calbindin+ naked axon. C, D) Remyelination was evaluated by counting either PLP+myelinated internodes or Caspr+ paranodal structures. Scale bar= 10µm

Cellular expression of BTK and effect of BTKi on remyelination in Xenopus brain

Cellular expression of BTK in the Xenopus and dose response of remyelination potency of BTKi : A-F) Immunodetection of BTK in microglial cells, but not in oligodendrocytes. Coronal(A-C) or horizontal (D-F) tissue sections across the brain stem off stage 52-53 MBP-GFP-NTR tadpoles doubly labeled with anti-BTK (A, C, D, F, red) and anti-GFP (B, C, oligodendrocytes, green)antibodies or isolectin IB4 (E, F, microglia, white). Note in A-C the complete exclusion of the two labeling, illustrating absence of BTK in oligodendrocytes. D-F) In contrast, all BTK+ cells werealso IB4+. However, some IB4+ cells (white arrows) were not BTK+. G) Demyelination of stage 52-53 MBP-GFP-NTR tadpoles was achieved by 10 days exposure to metronidazole (10mM) in theswimming water. Tadpoles were then returned to normal water or water containing increasing concentration of BTKi (MSC2494528) for 3 days. Remyelination was assayed by counting thenumber of GFP+ cells per optic nerve on day 3 of the repair period. Treatment of tadpoles with BTKi (MSC2494528) at concentrations ranging between 50nM and 1µM improved remyelination upto 1.7-fold compared to spontaneous recovery (control) set as 1. (*p < 0.01; **p < 0.001). Scale bar in A-F =20µm

CONCLUSIONS

Altogether, our data show that BTK inhibition has a net effect on remyelination both ex vivo (rodent) and in vivo (Xenopus), which suggeststhat BTK inhibition represents a promising new therapeutic strategy to promote remyelination by targeting microglia. This activity would becomplementary to the well-established inhibition of B cell function and the recently described promotion of anti-inflammatory macrophagedifferentiation.

BTK

BTK

PLP-GFP BTK

PLP-GFP Calb Caspr

Ctr

l

Ctr

l

LP

CB

TK

i

Iba1 BTK S100b BTK CD11b p-BTK S100b p-BTK p-BTK BTK

GFP

IB4 Merge

Merge

A BC D E

A B C D

A B C

D E F

G H I J K L M

A B C

D E F

G