Axonal control of myelin sheath length

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the myelinated axons, when one of the rare ensheathments was formed, Nfasca was surprisingly not excluded from underneath the myelin segment. Along RB axons, although not myelinated, Nfasca did also not form clusters, but it stayed homogeneously distributed.

Different localization dynamics of Nfasca, clustering along some but not all axons, might be explained by axon intrinsic differences. First, RB neurons are normally not myelinated, so there is no need to already cluster nodal proteins as there are no nodes to be formed. This would imply that these dynamics should be seen along all unmyelinated neurons. In my thesis, I only focused on RB neurons as largely unmyelinated neurons, but it would be interesting to investigate the differences of Nfasca dynamics along other unmyelinated axons. If there is no clustering this would imply that Nfasca clustering plays a role during myelination and therefore support the hypothesis of axonal mechanisms regulating myelination.

Another difference is, that RB neurons are special, as they only occur in fish and amphibians (Lamborghini, 1987; Williams et al., 2000), and during development they are replaced by DRG neurons. Some studies suggested that RB neurons die early in zebrafish during development (Williams et al., 2000; Svoboda et al., 2001), but we and others (Knafo and Wyart, 2018) observed RB neurons also at later stages. The different dynamics could therefore also be explained by the different evolutionary origin.

At first, around 3dpf, Nfasca was homogeneously distributed, also in CoPA and CiD axons, then clusters did emerge and they became more pronounced when the neuron stayed unmyelinated. I followed these clusters over time and found them to be remarkably stable.

They only move due to body growth. Very rarely I observed new clusters being formed or existing clusters disappearing. The high stability, as well as the moving due to body growth, might be explained by cytoskeletal anchoring of these clusters, similar to nodes. Clustering of nodal proteins, like sodium channels or AnkyrinG, have been shown before (Kaplan et al., 1997); (Freeman et al., 2015). In vitro, the cluster, or prenode formation depended on AnkyrinG, whereas Neurofascin186 was dispensable (Freeman et al., 2015). It could well

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be that this is also the case for the Nfasca clusters I observed in vivo. However, as Neurofascin has an AnkyrinG binding motif, the Nfasca clustering could also be due to prior AnkyrinG clustering.

I hypothesized that these Neurofascin clusters could serve as growth barriers during developmental sheath growth, explaining the high probability of asymmetric growth. To test if the clusters serve as growth barriers and therefore determine node position, I imaged unmyelinated axons with clusters and followed them until they got myelinated. I correlated the node position with the pre-myelination cluster position and found that the majority of nodes were formed at position with prior clusters, indicating that sheaths had stopped growing once they reached the clusters. As mentioned before, a mechanism where the axon intrinsically defines the node position could explain how specific myelination patterns along axons (Ford et al., 2015) are implemented. There are some known factors by which axons regulate regions that are to be myelinated or to prevent myelination. JAM2 was shown to prevent somatodendritic myelination (Redmond et al., 2016), and it was has been shown that Galectin-4 expression in neurons labels unmyelinated stretches and prevents their myelination (Díez-Revuelta et al., 2017).

When I followed the axon in these experiments I did not always see clusters forming. Often the axons did show homogeneous distribution and at the next imaging timepoint they were fully myelinated. The clustering and myelination could happen almost simultaneously, making it hard to disentangle them and assess which of them happens first. To circumvent this problem, I aimed to inhibit myelination to investigate if clusters still form in the absence of myelin. When doing so, by the ablation of NTR expressing OPCs with MTZ, I still observed a substantial amount of myelin formed in the beginning. The NTR was expressed under control of olig1 promoter elements and when these cells emerge it takes some time for the NTR to be expressed and to function. The formed myelin might therefore be explained by the OPCs that had already differentiated before the NTR could transform MTZ into the DNA crosslinking agent. Instead of a prevention, myelination was delayed.

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Nevertheless, it prolonged the time in which the neuron was unmyelinated, and I could see clusters forming. When the axons were getting myelinated and nodes formed, I assessed the cluster node correlation as well and found similar results. I could see that the majority of nodes had formed at positions with pre-myelination clusters, suggesting that clusters do emerge before myelination and are predictive for node positioning.

If Neurofascin clustering plays an important role in defining node position, it would be interesting to investigate what happens when Nfasca is knocked out. If it has a role in determining node position and stops sheath from growing, in Nfasca KO more sheaths should grow symmetrically and they should also be longer.

Based on the experimental evidence I, hypothesize that the axon determines node of Ranvier position, however, the exact pattern might be refined by the communication of myelin sheaths/oligodendrocytes and the ensheathed axon. The phenomenon of pre-patterning with subsequent refinement of the pattern has already been suggested in other systems, like nicotinic acetylcholine receptor clustering at the motor end plate (Kummer et al., 2006).

If the axon determines the node position, how should the sheaths then know how fast they should grow, as the growth rates determine sheath length differences in the second growth phase? One possible explanation could be, that there are gradients of either permissive or repulsive cues that regulate growth rates locally at the axon-glia interface.

In the last experiment of my thesis I aimed to test for axon intrinsic control of sheath length.

To investigate that, I wanted to change an axon intrinsic feature and test for node of Ranvier remodeling. I therefore manipulated activity by optogenetics and hyperactivated a spontaneously very silent neuron and followed node position over time. With pre-stimulation imaging I secured that the sheaths have reached their elongation phase. Upon the optogenetic stimulation I observed that some nodes, and therefore also sheath lengths, remodeled and changed their relative position. Some sheaths showed deviations from their predicted length, indicating activity dependent remodeling. However, I could observe similar

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effects in my control experiments, where I did not express Channelrhodopsin. This ‘off-target’ effect might be explained by the stimulation of the fish with bright blue light. I observed increased swimming in the light stimulated fish, which could also indicate an overall increased neuronal activity, making it impossible to disentangle the Channelrhodopsin mediated effect from the general light stimulation effect. Furthermore, it has recently been shown that stimulation with blue light, in contrast to green or red light, alters neuronal gene expression (Tyssowski and Gray, 2019), adding another layer of uncontrolled effects. Hence, we decided, for our future experiments, to switch from Channelrhodopsin to the light activated channel ChrimsonR (Klapoetke et al., 2014).

ChrimsonR is activated by red light, and therefore not altering gene expression (Tyssowski and Gray, 2019), and it is more light sensitive than Channelrhodopsin, which should reduce the ‘off-target’ effect by the light stimulation.

Nevertheless, activity induced remodeling could also be caused by axon to oligodendrocyte communication. Local vesicle release or other mechanisms, as a consequence of increased activity, could induce sheath remodeling. One way of testing for that, excluding any glial influence, would be to test if pre-myelination clusters of Nfasca can remodel. As mentioned before, without interference these clusters are remarkably stable, probably due to anchorage to the cytoskeleton. Activity dependent changes in cluster position would therefore imply that the axon intrinsically regulates where these clusters are deposited along the axon and, as the clusters are determinants for nodes, also where nodes of Ranvier will be.

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