Investigating the mechanism of action underlying the ability of kappa opioid receptor agonists to promote oligodendrocyte differentiation and myelination in vitro

These side effects are believed to be mediated by KOR signalling via β-arrestin 2 signalling pathways. In contrast, KOR activation of G-protein signalling pathways is typically associated with therapeutic effects with reduced side-effect profile. Structurally modified G-protein biased KOR agonists can be developed into safe remyelinating therapeutics.

Therefore, this thesis aimed to identify and investigate the mechanism of action of novel compounds from our library of KOR agonists (sourced from our collaborator Professor Prisinzano) that can enhance and stimulate OPC differentiation and myelination in vitro. This study used in vitro OPC-containing cultures from the whole brain of 1-7 day old C57BL6 mice and medium throughput confocal screening assays to evaluate the ability of novel KOR agonists to promote OPC differentiation and myelination.

In our OPC-containing cultures, KOR agonists [U50,488; U69,593; LDK93; LDK95; LDK276; sal A; mesyl sal B; EOM sal B; βTHP sal B; 16-ethyl sal A; 16-bromo sal A; nalfurafine; tifluadom; LDK376 and triazole 1.1] stimulated OPC differentiation into mature OLs. Furthermore, these OPC differentiation effects were KOR mediated as the KOR antagonist, nor-binaltorphimine, prevented KOR-induced OPC differentiation.</p><p>In order to identify the specific cell signalling pathways underlying KOR’s ability to differentiate OPCs, we used specific inhibitors of known KOR signalling, including ERK1/2, mTOR, p38 MAPK and JNK pathways.

The number of mature OLs following co-treatment with ERK1/2 inhibitor, LY3214996, and KOR agonists [U50,488; LDK276; nalfurafine; EOM sal B; 16-ethyl sal A] was significantly lower compared to vehicle-treated cultures. Similarly, co-treatment with the mTOR inhibitor, rapamycin, and KOR agonists [U50,488; LDK276; nalfurafine; EOM sal B; and 16-ethyl sal A] reduced the number of mature OLs. These results suggested that KOR agonists U50,488, LDK276, nalfurafine, EOM sal B, and 16-ethyl sal A mediate OPC differentiation via ERK1/2 and mTOR pathways.

Furthermore, co-treatment with the p38 MAPK inhibitor, SB203580, and KOR agonists [U50,488; LDK276; and EOM sal B] reduced the number of OLs. This suggested that KOR agonists promoted OPC differentiation through the p38 MAPK signalling pathway. KOR agonists were further assessed for their potential to differentiate OLs that can wrap myelin in an in vitro myelination assay.

In this assay, aligned polycaprolactone (PCL) nanofibers were utilised to investigate the effects of KOR agonists (U50,488; LDK276; 16-ethyl sal A and nalfurafine) in promoting myelination. The effect of each KOR agonist was quantified by measuring the area of mature OLs and number of myelin segments wrapped by mature OLs on nanofibers. KOR agonist treatment significantly increased the area, and number of myelin segments in mature OLs compared to vehicle-treated OLs.

This suggested that KOR-mediated mature OLs have the ability to wrap myelin sheath around nanofibers and they promoted myelination. Among all KOR agonists evaluated, treatment with LDK276 and 16-ethyl sal A significantly increased the number of mature OLs, concurrently increasing the area and branch complexity of OLs. Moreover, LDK276 and 16-ethyl sal A treated OLs showed an increased number of myelin segments.

The present study concluded that LDK276 and 16-ethyl sal A are the most effective compounds at promoting OPC differentiation and myelination. A clinically approved KOR agonist, nalfurafine, has also shown promising results in our myelination assay. This is the first study to investigate the effects and mechanism of action of wide-ranging novel structural KOR agonists in promoting OPC differentiation and myelination.

Overall, the findings in this thesis identify KOR agonists (LDK276, 16-ethyl sal A and nalfurafine) as the best, promising, potential therapeutic candidates in the development of remyelination-based therapies for demyelinating diseases such as MS.</p>