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 byimmunolabeling in normal adult mouse brain tissue sections and organotypic cerebellar slicecultures 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. Aigrot1, E. Martin1, 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, Paris, France, 2EMD Serono Research & Development Institute, Inc., Billerica, MA, United States, 3AP-HP, Saint-Antoine Hospital, Paris, France, 4AP-HP, Pitié-Salpêtrière Hospital,

Paris, France, 5Ares 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 ofPLP+ internodes and the number of Caspr+ paranodal structures in cerebellar slices from plp:GFP mice treated with aninhibitor 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

Co

nt

LP

C

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-labellingexperiments confirmed that BTK-positive cells were observed largely in Iba1+ microglia (73.1%), as well as in a smallfraction of S100+ astrocytes (25.6%), with minimal labelling in CC1+ oligodendrocytes (<1%). BTK was not detected inNeuN+ neurons nor in Olig2+/CC1- oligodendrocyte precursor cells. C) Higher magnification illustrating co-localisation ofBTK and p-BTK in microglia and astrocytes

A B

C

BTK

BTK

GFP

GFP

D0

D1

0

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

RESULTS

I NTRODUCTI ON

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

CONCLUSI ONS

• 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.

TI MEPOI NTS :

• 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

SWI MMI NG BEHAVI OR I S ALTERED FOLLOWI NG DEMYELI NATI ON

VI SUAL AVOI DANCE PERFORMANCE DECLI NES

AFTER DEMYELI NATI ON

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

METRONI DAZOLE- I NDUCED CONDI TI ONAL DEMYELI NATI ON I S FOLLOWED BY SPONTANEOUS REMYELI NATI ON

MBP GFP Nitroreductase

We have developed a transgenic Xenopus laevis permitting live imaging of demyelination and remyelination by conditionallytriggering 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) regulatorysequence. The NTR enzyme converts the nitro radical of prodrugs, such as metronidazole (MTZ), to a highly cytotoxichydroxylamine derivative. In MTZ-exposed transgenic MBP-GFP-NTR tadpoles demyelination is restricted to the CNS, withoutaxonal 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. Afterdemyelination there is a clear increase in the level of expression of BTK, which is mostlyexpressed 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 newtherapeutic 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) andat the end of metronidazole treatment (D10). We observed an increase in the number of BTK+ cells under demyelinatedconditions (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 therepair period. Of note BTKi (MSC2494528) treatment resulted in 1.7-fold improvement of remyelination compared tospontaneous 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 multiplesclerosis, microglia are the Janus of the innate immune response, exerting either aproinflammatory 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 cellreceptor, myeloid activation via Fc receptors, and differentiation of proinflammatorymacrophages in response to GM-CSF in vitro. However, the role of BTK in CNS, especially inCNS glia, is unknown.The aim of our study was to examine the cellular expression of BTK in the CNS and toinvestigate the consequences of BTK inhibition on remyelination both ex vivo and in vivousing 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