Investigating mechanisms of oligodendrocyte precursor cell recruitment in zebrafish models of myelin development, plasticity, and repair

Abstract

Myelination is an essential process for the function of the central nervous system (CNS), enhancing signal conduction velocity and providing trophic support. The CNS becomes increasingly myelinated over long periods of time, lasting decades in humans. Furthermore, new myelin can form in response to experience/neuronal activity and plays a role in learning. This process is referenced as adaptive myelination or myelin plasticity. On the other hand, new myelin also forms in response to previous myelin damage and oligodendrocyte loss, as it occurs in diseases such as Multiple Sclerosis, a process termed remyelination. As new myelin can be generated in these different contexts, it is important to understand myelination mechanisms in health and disease.

Myelination is carried out by oligodendrocytes wrapping their plasma membrane around axons and forming insulating sheaths. Myelinating oligodendrocytes differentiate from a population of specified precursors, termed oligodendrocyte precursor cells (OPCs). OPCs are an abundant, electrically excitable cell type that tiles the central nervous system and exist lifelong. The constant presence of OPCs which have the potential to differentiate allows myelination to continue throughout life. Although new oligodendrocytes increasingly differentiate from the pool of OPCs in response to CNS activity and damage, it is not known whether the mechanisms driving this process are the same. This is important to understand in order to properly enhance remyelination in demyelinating diseases like Multiple Sclerosis.

The aims of my PhD research were therefore: 1. to investigate the relationship between neuronal and OPC activity in healthy CNS development, 2. to investigate how neuron and OPC activity change in demyelination, 3. to establish a model for activity-enhanced oligodendrogenesis and 4. use this model to determine if different OPC subgroups are recruited in development, myelin plasticity and remyelination.

To approach these aims, I used zebrafish larvae as a model organism. Zebrafish are a small vertebrate species with optical clarity at larval stages, with many genetic tools available to easily visualise/manipulate different cells in the system. Thus, they can be used for live imaging while intact, and therefore to observe neuron and OPC behaviour in vivo with high resolution.

For my first aim, I established a methodology of dual colour calcium imaging, which allowed me to measure endogenous changes in activity of neurons and OPCs simultaneously. I found that approximately one quarter of OPC microdomain transients occurred following a proximate glutamatergic or GABAergic axonal event, with no difference between these two neuron classes. My results indicate that neuronal activity may drive some of the OPC calcium responses in healthy conditions, but that a high proportion of OPC calcium signalling occurs independently of direct neuronal signalling.

For my second aim, I investigated changes in calcium signalling activity in neurons and OPCs in a zebrafish model of experimental demyelination. While mRNA staining of early activity marker cFos showed a higher number of active neurons following demyelination, calcium imaging showed that firing frequency and amplitude of glutamatergic and GABAergic neurons was unaffected by demyelination. However, demyelination led to an enhanced synchrony of GABAergic axons and cell bodies, further indicating that neuronal calcium activity is somewhat affected in demyelination. Furthermore, OPC calcium signalling was enhanced during an active remyelination period, as the number of active process regions increased. Given that activity-enhanced OPC recruitment has also been shown to be mediated by enhanced OPC calcium activity, these data suggest that mechanisms of OPC recruitment may converge on enhanced OPC calcium responses.

In my third aim, I describe the development of several models to enhance oligodendrogenesis in response to selective neuronal activation. An optogenetic stimulation protocol was successfully developed to enhance specifically neuronal activity and swimming behaviour, which was scalable and tolerable in a chronic stimulation paradigm. However, this protocol did not robustly increase oligodendrogenesis. In contrast, a protocol to enhance swimming activity by chronic exposure to moving gratings that drives optomotor response led both to enhanced activity as well as increased oligodendrogenesis, establishing it as a model of adaptive myelination to use in further experiments.

Lastly, for my fourth aim of investigating OPC cellular fates in different oligodendrogenesis conditions, I investigated the recruitment of previously established OPC subgroups in models of enhanced activity and demyelination. When examining the morphology of OPCs recruited in development, adaptive myelination and demyelination, I found that the subpopulation of OPCs that was mostly recruited to myelination was the same, implying again that these mechanisms converge across conditions. However, when I labelled OPCs with a subpopulation-specific marker Gpr17, differences in the recruitment mechanisms emerged. While the proportion of OPCs expressing Gpr17 remained the same between healthy development and enhanced activity, this proportion was enhanced following demyelination.

In conclusion, my results show that OPC recruitment in adaptive myelination and myelin repair converges on multiple steps, such as enhanced OPC calcium and enhanced differentiation of mostly “myelinating OPCs”. However, my results also reveal differences in OPC behaviour between healthy conditions and repair, with the enhanced recruitment of a Gpr17-expressing OPC subpopulation following demyelination. Therefore, when trying to enhance remyelination in a disease setting such as Multiple Sclerosis, the cellular targets might have to be myelin regeneration specific, instead of attempting to recreate developmental or activity-driven myelination.