Investigating myelination and remyelination in zebrafish

Abstract

Central nervous system (CNS) myelination is important for proper nervous system function in vertebrates. In demyelinating diseases such as multiple sclerosis, autoimmune-mediated myelin destruction results in neurological impairment; and although remyelination does occur spontaneously, it is poorly understood and insufficient in humans. Zebrafish (Danio rerio) are known to harbour tremendous regenerative capacity of various CNS tissues; however, there is presently only little knowledge of their myelin repair efficiency. An experimental model of myelin injury in zebrafish would permit study of the mechanisms involved in successful remyelination and could potentially guide the development of novel therapeutic agents for mammalian remyelination. This doctoral thesis describes the characterisation of the novel myelin protein Claudin k in zebrafish, demonstrates the establishment of adult zebrafish as an experimental model for CNS de- and remyelination and explores some mechanisms underlying myelin repair. A variety of myelin markers have previously been investigated in zebrafish, including myelin basic protein and myelin protein zero. However, the use of these is limited by either late developmental expression or presence in compact myelin only. Claudin k is a novel tight junction protein specific to zebrafish CNS and PNS, which can be observed early in development and throughout nervous system regeneration. Utilising specific antibodies and a novel transgenic zebrafish line, in which the claudin k promoter drives the expression of green fluorescent protein in myelinating cells, the studies herein characterise the expression of Claudin k, demonstrate the fidelity of the transgenic construct, and investigate the relationship of Claudin k with established myelin and CNS inflammation markers. Data demonstrate that Claudin k expression closely resembles expression patterns of the endogenous gene, and as such provides a key tool for examining CNS myelination in zebrafish. For the study of de- and remyelination in the zebrafish, the experiments herein describe the use of lysophosphatidylcholine (LPC), a detergent-like myelin toxin, which is used widely in rodent models to demyelinate axons. Its application to the adult zebrafish optic nerve induced focal demyelinating lesions, critically without detectable axonal injury, and permitted the study of time course and efficiency of remyelination. Myelin in the lesion area was reduced as detected by both immunohistochemistry and electron microscopy at 8 days post lesion (dpl), and return of the markers to control levels suggested regeneration by 28 dpl. In addition microglial activation was observed along the optic pathway, which also returned to levels compared to unlesioned control by 28 dpl. In young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres showed no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. However, in old fish (aged 18+ months), remyelinated fibres presented with thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. While the zebrafish as an experimental system has tremendous benefits, such as potential for drug screens using the transparent larvae, capacity for transgenesis and live imaging, experimental models in zebrafish potentially bear several limitations, in particular their distant relationship to humans. To determine whether zebrafish remyelination involves homologous signalling mechanisms to mammals, demyelinated zebrafish optic nerves were treated with human recombinant Semaphorin 3A, an axonal guidance molecule which is well known to inhibit oligodendrocyte precursor cell (OPC) recruitment and remyelination in mammals. Results demonstrated fewer oligodendroglial cells at 14 dpl and less myelinated fibres at 28 dpl in the optic nerve lesion area compared to control treated animals, supporting the hypothesis that zebrafish remyelination may indeed respond to human signalling molecules. Taken together, the findings in this doctoral thesis suggest that this new experimental zebrafish-based model of CNS remyelination can be added to the suite of current models to better understand the remyelination process and that some signalling mechanisms observed in mammals around myelination and OPC recruitment are likely conserved in the zebrafish. In addition, it could potentially be used to discover novel therapeutic targets that promote myelination in injury.