Imaging the assembly, disruption and restoration of nodes of Ranvier in myelinated nerves

Oligodendrocytes are the cells in the human brain and spinal cord (central nervous system) that produce the myelin sheath around nerves. Schwann cells do the same job in the rest of the nervous system (peripheral nervous system). These cells myelinate nerve fibres by extending processes which encircle the nerves many times to form a multi-layered sheath. Each nerve fibre has many myelin segments and in humans each segment can be over a millimeter in length, with gaps between the segments of about a thousandth of a millimeter. Myelin promotes rapid communication not only between different groups of nerve cells within the central nervous system, but also between nerve cells and muscles. This is because wrapping myelin around nerves causes specialized pores at the nerve surface called sodium channels to become concentrated in the gaps between successive myelin wraps. These gaps are called nodes of Ranvier and their high concentration of sodium channels is crucial for the rapid electrical conduction of nerve impulses.

When myelin is destroyed, as in multiple sclerosis (MS) in the central nervous system, or Charcot-Marie-Tooth (CMT) disease in the peripheral nervous system, we lose functions for two reasons. First, the speed of nerve conduction slows because sodium channels diffuse away from the node, and secondly, without a myelin sheath the nerves start to degenerate. It is believed that nerve degeneration is linked to the disruption of the nodal sodium channels, and their dispersion is also believed to be at the root of other distressing aspects of these diseases such as pain. Demyelinating diseases are relatively common: there are about 80,000 people in the UK with MS and about 30,000 with CMT. Thus far there are no cures and no therapy halts disease progression.

There has been considerable progress in identifying the accessory proteins that persuade sodium channels to go to the node and remain there, primarily by knocking the genes that encode the proteins out in mice. This is how we discovered a group proteins that are encoded by a gene called Neurofascin which have a key role in "clustering" sodium channels. However, we have no idea how they do it. Therefore, two key question need to be answered. The first is, how does myelination cause Neurofascin proteins to assist in concentrating sodium channels at nodes of Ranvier? Secondly, why does loss of myelin cause sodium channels to disperse?

We are planning to answer these question by studying myelination in living tissue using very advanced microscopical techniques which have been recently developed and for which Edinburgh University is now very well-equipped, thanks to major funding from the Medical Research Council (MRC). It is now possible to observe the key nodal proteins once they have been labelled with a fluorescent tag and identify their location and movement over periods from hours to days in living tissue in which myelination and demyelination can be observed.

We believe that we will gain important new insights into the behavior of these key proteins of the node of Ranvier which will allow us to determine what happens to sodium channels in demyelinating disease and why. Since there is continuing interest in sodium channels as therapeutic targets in demyelinating disease to either limit the degeneration of nerve fibres and/or mitigate distressing symptoms such as pain, we believe the outcomes of this work will be of great interest to those who are attempting to develop such drugs; indeed it may be that such drugs might be more usefully targeted towards the proteins that interact with sodium channels, such as the Neurofascins.

The purpose of this project is to use advanced optical microscopy and live imaging to reveal how fluorescent proteins associated with voltage-gated sodium channels (Nav), including Neurofascin186, are trafficked to the node of Ranvier during myelination, how they behave once there and how their localization and dynamics are affected by demyelination and remyelination in the CNS.
We have generated transgenic mouse lines shown to be suitable for live fluorescence imaging in collaborative pilot experiments with Prof. T. Misgeld, Technical University Munich. A fluorescently-labelled derivative of saxitoxin developed for live imaging of Nav by Dr Justin Du Bois, Stanford University will be supplied to us which enables real-time imaging of native Navs in live cells at the single-molecule level.
Nodes are approximately 1 micron wide but spatial resolution in conventional optical microscopy is ultimately limited by the diffraction of visible light (250 nm). Several techniques have been invented to achieve higher spatial resolution to <50 nm collectively termed super-resolution microscopy which offer the opportunity not only of visualizing much finer detail within nodes but also of providing quantitative data on mobilities, dynamic behaviours, interactions and spatial patterning of individual molecules during myelination, demyelination and remyelination.
Two recent awards by MRC for optical imaging infrastructure in Edinburgh now make this work feasible (£2M, ESRIC, 2013, MR/K01563X/1; £1.7M, 2013: Ref. MR/K015710/1). Prof. Rory Duncan is PI and Director of ESRIC and will be our collaborator in super-resolution microscopy.
We believe that at the conclusion of this project we will have a much clearer view of nodal protein dynamics as they occur in living tissue; further, by applying these approaches to a genetically tractable model we expect to gain new insights into how the targeting and dynamics of these proteins are influenced by demyelination and repair in the CNS.

MS, the subject of an MRC Highlight Notice, is the commonest neurological disease affecting young adults with a prevalence of approximately 0.15% in the UK. Health economists have variously estimated the annual cost of MS in the UK at between 1.4 and 3 billion pounds. Clearly, developing effective therapies is a matter of urgency due to both the human and economic costs. Inherited peripheral demyelinating neuropathies of the Charcot-Marie-Tooth type with a prevalence of approximately 0.04% represents one of the commonest inherited neurological disorders.

Nevertheless, it is difficult to identify how progress will be made, particularly in the area of neuroprotection, without significant progress in our understanding of the fundamental biology of myelination and demyelination.

Sodium channels have already been considered as targets for drugs that might promote neuroprotection in MS. Indeed Lamotrigine has been the subject of a phase II clinical trial. Although shown to be only marginally effective, it is now being tested in combination with other drugs such as beta-interferon. Clearly, our understanding of how manipulating sodium channels might promote axonal survival in myelinated nerves lags well behind the enthusiasm for clinical trials using drugs that might influence sodium channel function, stability and trafficking.

Our innovative research programme focuses on the development of integrated multi-dimensional imaging promising a step change in our understanding of the molecular organization of the node of Ranvier, a crucial structure in myelinated nerves, in health and disease.

The planned programme of research and innovation focuses on the development of innovative biomedical research which will have broad relevance to diseases of the nervous system and is thus consistent with MRC's strategy -'Changing Lives'- by ultimately delivering benefits to patients and their carers. The knowledge gained from addressing sodium channel clustering will be of immediate interest and benefit to industry and scientists researching the initiation and progression of disease. Details of technical innovations related to instrumentation will be of interest to companies who manufacture and develop optical microscope systems, and components.

To ensure information generated during the course of the planned programme of work achieves maximum reach within the biomedical community we will ensure all data is preserved and that image datasets and their analyses are made available on publicly accessible web-sites. We anticipate that the new knowledge of the biology of sodium channel and associated protein trafficking will be of long-term benefit for strategies in MS treatment, and indeed also in demyelinating diseases of the peripheral nervous system.

Our plan of work will benefit tremendously through interaction with Prof. Misgeld of the Technical University Munich, who is a world leader in live imaging in the mammalian nervous system. Hence the project offers opportunities for training and capacity building in biomedical research and technologies related to image acquisition and analysis that will have a long-term benefit on UK-based neuroscience and ultimately on health and wealth creation. On the other hand promising research will provide leverage with policymakers in order to ensure that efforts to improve neurological disease are increased in a manner that is in line with the ever-increasing burden of such diseases on the nation's wealth.