Phd in CLINICAL EXPERIMENTAL NEUROSCIENCE AND PSYCHIATRY

Thesis title: BTK inhibitors modulate remyelination in Multiple Sclerosis

Background: Myelin repairing mechanisms in people with MS are altered and demyelinated axons are exposed to stressors, leading to neurodegeneration especially in progressive MS. Nowadays, remyelination is a therapeutic unmet need in MS. Bruton’s tyrosine kinase inhibitor (BTKi), such as Ibrutinib, are thought to be able to delay MS progression. To date, remyelination has been tested in vitro using prenatal models. Here, we test Ibrutinib effects on remyelination proposing a novel in vitro model using cells obtained from post-natal mice. Firstly, we verified that cultures by post-natal mice remyelinated after LPC-mediated demyelination. Then we assessed that Ibrutinib improved remyelination in mouse model of de- and re-myelination. It is known that Ibrutinib effects require a crosstalk between BTK-expressing cells and OPCs. We evaluated extracellular vesicles (EVs) release from microglial cells but no differences in quantity release was observed. We hypothesize that OPCs/microglia interaction lays on EVs content modification induced by Ibrutinib. This is the first study showing the BTKi positive effects on remyelination in an animal in vitro model allowing us to quantify remyelination modulated by treatments and to reduce animal sacrifices to pursue the 3R. Aims: 1) To validate an in vitro model of de- and re-myelination using post-natal mice; 2) To investigate Ibrutinib effects on a remyelination in vitro model; 3) To evaluate Ibrutinib action mechanism on target cells (monocyte-macrophage lineage). Materials and Methods CELL CULTURES – Mixed cell cultures were obtained by spinal cord of pups. Cultures have then been exposed to lysophosphatidylcholine (LPC) causing demyelination. Cells were cultured for additional 7 days. Ibrutinib (1 µM) was added for 7 days. IMMUNOFLUORESCENCE – Cultures were fixed and exposed to permeabilizing and blocking solutions. Cover slips were then incubated overnight with primary antibody solutions and, consequently, with fluorescein-labeled secondary antibody solution and Hoechst solution (1:1000). Myelination index was computed as a percentage ratio between number of myelinated axons and total axons. Results 1) Characterization of mouse spinal cord cell cultures from mouse pups: Immunofluorescence analysis showed that cultures were constituted by about 45% OPCs/Oligodendrocytes, 25% neurons, 20% microglia, 10% astrocytes. In postnatal cultures myelination percentage reached 60%. LPC induced demyelination after 24 hours. After 7 days, a process of remyelination was observed. After LPC-mediated demyelination, myelination percentage improved (remyelination) even if not reaching basal starting levels. 2) Ibrutinib induced complete recovery of myelination after LPC damage: Myelination percentage in Ibrutinib-treated remyelinated cultures were superior to Ibrutinib-untreated remyelinated cultures and similar to basal levels obtained by undemyelinated controls. 3) Ibrutinib modulated microglia-derived EVs content by increasing mir-223 expression levels: To understand the mechanism of action underlying Ibrutinib-mediated effect, extracellular vesicles (EVs) release from microglial cells (BV2 cell line) was evaluated showing no differences in quantity between untreated and Ibrutinib-treated cells, as reported in graph below. Since in MS several inflammation- and remyelination-associated microRNAs (miRNAs) have been identified as dysregulated, EVs content was examined and an increase in mir-223 expression level was observed in Ibrutinib-treated BV2 cells when compared to ctrl. Discussion This is the first study showing the BTKi positive effects on remyelination in an animal in vitro model. Ibrutinib effects require a crosstalk between BTK-expressing cells and OPCs. We evaluated extracellular vesicles (EVs) release from microglial cells but no differences in quantity release was observed. We hypothesize that OPCs/microglia interaction lays on EVs content modification induced by ibrutinib. Conclusions - Cultures obtained by postnatal mice showed to be able to remyelinate after LPC-mediated demyelination; - Ibrutinib improved remyelination in mouse model of de- and re-myelination; - Ibrutinib action mechanism seems to involve the modulation of EVs release by target cells