Molecular mechanisms of voltage-gated potassium channel clustering in myelinated axons

The communication between our sense organs, such as our eyes or skin, and our brain and spinal cord occurs through electrical signals that are carried by our nerves. Nerve are composed of bundles of neuronal extensions called axons. Axons conduct these signals through the regulated flux of Sodium and Potassium ions over the axonal membrane through specific channels. Many of the axons in our nerves are insulated by a fatty layer called myelin which is made and maintained by support cells called Schwann cells and oligodendrocytes. The myelin layer speeds up nerve signaling enormously, allowing us to respond rapidly and with great accuracy to any physical challenge coming our way. The way myelin speeds up signal conduction is by clustering different ion channels to high density at regularly spaced regions along the axon.

The importance of this arrangement and density of ion channels becomes apparent when the integrity of the myelin layer is damaged as in multiple sclerosis or in diabetes, which alters or even block conductance. But how these ion channels are arranged and kept in specific regions is largely unknown.

In this work we will examine the molecules that drive the clustering of one type of ion channel; the Kv1 class of potassium channels. Kv1 channels play important roles in normal nervous system function and mutations in the genes that encode these proteins give rise to neurological diseases such Ataxia and epilepsy. These channels are found in very high density in myelinated axons but also in other parts of the neuron.

We have discovered that the specific positioning of these channels in myelinated axons is a function of two proteins; the ADAM23 receptor and its soluble binding partner LGI3. We will investigate how these molecules assemble the Kv1 channels and keep them in their fixed position. We will identify what other proteins are involved in this process and we will determine how conduction properties of myelinated axons is altered when Kv1 channels are mis-localised or removed from the axonal membrane. These important insights will further our understanding of how Kv1 channels are distributed in myelinated axons and inform us about the mechanisms that regulate Kv1 channels in other parts of the neuron to affect the basic physiological properties of the neuron and the neuronal networks they participate in, and lead to potential improvements in treating human ataxic and epileptic disorders.

Shaker-type voltage-gated potassium (Kv1) channels play crucial roles in neuronal excitability, high frequency firing and neurotransmitter release. Their function does not only depend on their intrinsic biophysical properties but also on the exact cellular localization and density.

We discovered that the high-density accumulation of Kv1 channels in myelinated axons is dependent on ADAM23 and its ligand LGI3, challenging the long-held dogma that this accumulation is a function of the interaction between the cell adhesion molecules CASPR2 and TAG1.

We hypothesis that ADAM23 drives the assembly of juxtaparanodal Kv1 clusters through an LGI ligand dependent mechanism.

ADAM23 belongs, together with ADAM22 and ADAM11, to a subgroup of the larger family of integrin receptors and metalloproteinases that are involved in diverse developmental processes including cell migration, axonal path finding and processing of growth factors.
LGI3 is a member of a small family of four closely related proteins implicated in synaptic plasticity and epilepsy.

In this proposal we aim to establish the exact developmental accumulation of Kv1 channels and how this process is affected by LGI-ADAM23 interactions and whether this mechanism involves an interaction with CASPR2 and ADAM22 (AIM1). We will identify ADAM23 (and ADAM22) interacting proteins using our unique set of transgenic animals and in vivo BioID technology (AIM2) and establish the functional role of these interacting proteins through CRISPR-Cas9 mediated genome editing in myelinating neuron-Schwann cell co-cultures (AIM3).

The mechanistic insight gained through this work will have wider implications for our understanding of ADAM-LGI functions in Kv1 biology in other cellular compartments such as the synapse and axon initial segment in the healthy and diseased nervous system, and have the potential to improve treatments for human ataxic and epileptic disorders.

This research will have considerable impact in several areas.

Scientifically
This research challenges long held ideas about the molecular mechanisms through which Kv1 channels accumulate in myelinated axons as it provides novel conceptual insights into the way LGI and non-metalloproteinase ADAM proteins regulate Kv1 channel distribution in neurons. Moreover, our work sheds light on the specificity of LGI:ADAM interactions in myelination and ion channel biology.

Social-economic
Alterations in Kv1 channels, either through genetic mutation or immune-mediated, cause ataxia, epilepsy, spastic paraplegia and memory disfunction. It is expected that the social burden associated with these diseases will only further increase in the aging population and current treatments are limited. Insights into Kv1 biology have the potential to improve treatments for these human neurologic disorders and improve quality of life.

Technologically
Our work furthers the development of the BioID technology as it takes it firmly into in vivo applications using transgenic mice and increases the capacity of our analytical repertoire. At the same time, we will adopt and validate CRISPR-Cas9 genome editing technology in in vitro neuron-Schwann cell co-cultures to further reduce the number of experimental animals used in this study, thus contributing directly to the 3Rs.

Collaboratively
We have three important collaborators in this program that bring a wide range of expertise to the project. The technical expertise in mouse genetics, myelin cell biology and BioID technology in combination with the electrophysiology of sensory processing expertise of my co-investigator Dr Carole Torsney is essential to the successful execution of this project. Furthermore, we benefit from the great expertise and advice from our collaborator Prof Elior Peles and from mass spectrometry service and advice from Prof Rapsilbers.

Training and Teaching
At the end of this project we will have trained one research assistant, two PhD students and at least three MSc students. Through our teaching in diverse neuroscience programs, this research directly benefits students at all levels of academic training.