Investigating the assembly of axonal domains for saltatory conduction

Abstract

The assembly of functional axonal domains including the axon initial segment (AIS) and nodes of Ranvier in myelinated fibres are essential in promoting nerve impulse conduction in the vertebrate central and peripheral nervous system (CNS and PNS). We still do not fully understand what determines exclusive axonal selection by myelin-forming glia, nor do we understand the mechanisms that determine myelin sheath length. Further, although progress has been made in understanding how the AIS is assembled in central neurons, little attention has been paid to the assembly and maintenance of the AIS-like domain, the Proximal Segment (PS), in peripheral Dorsal Root Ganglion (DRG) neurons.

Neurofascin B (NfascB), a zebrafish cell adhesion molecule homologue of the murine glial protein Neurofascin155 (Nfasc155), prevents aberrant myelination of neuronal cell bodies in the CNS. Moreover, NfascB and its axonal partner, Caspr, influence myelin sheath length. I have found using a combination of knock out and transgenic mice, that the roles of Nfasc155 and Caspr in myelin targeting and in determining myelin sheath length are conserved in the CNS of mammals.

Next, I focused on investigating the assembly and maintenance mechanisms of PSs in PNS DRG neurons. First, I established the existence of PSs in vivo. Subsequently, by using DRG neuronal cultures, I found that similar to AIS assembly, PS assembly does not require myelination; nevertheless, myelinating conditions do influence DRG neuron morphology leading to a higher proportion of bipolar and pseudounipolar neurons. Furthermore, I addressed the role of the neuronal Neurofascin isoform, Nfasc186, in PS assembly and maintenance. I found that similar to the AIS, Nfasc186 does not play a role in PS assembly. However, as opposed to the AIS, Nfasc186 does not play a role in PS maintenance.

To address how membrane proteins such as Nfasc186 are delivered to the PS, I used Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) on live DRG neuronal cultures. I found that Nfasc186 is delivered by bidirectional lateral diffusion from soma and distal axon to the PS of PNS DRG neurons, the same pathway followed for the delivery of Nfasc186 to the AIS of CNS cortical neurons.