Neuronal Logistics

Brain cells are uniquely shaped among the many cell types of the body. While most cells are more or less rounded or square-shaped, neurons grow one or more long axons that can reach lengths of a meter or more. To keep these axons alive and functional, neurons are dependent on an intracellular transport system, along which cargoes are carried from the cellbody to the far edge of the cell and back again. This transport duty is performed by motor proteins which bind and through conformational changes can move along microtubules. The purpose of this thesis is to contribute to our knowledge of this transport system and its role in the development and function of the central nervous system, with a special focus on the role of this system in multiple sclerosis (MS).

This thesis can be divided in three separate parts. The first part focuses on the role of transport in development, first on the level of individual neurons and neuronal networks (chapter 2), then in the development of the cerebellar cortex (chapter 3). In the second part we examine the role of transport in MS (chapters 4-6). Finally, in the third part we demonstrate how the application of superresolution microscopy can contribute to a better understanding of the transport system (chapters 7-8). In chapter 2 we descibe the contribution of the neuronal transport system in the development and maintenance of synapses. These contact points between neurons can be reinforced or degraded, altering the communication within a neuronal network. Through this mechanism, the brain is able to store new information and learn new skills. Both the microtubuli cytoskeleton itself as well as the transport taking place along the cytoskeleton is essential for synapic plasticity and therefore for learning and memory. In chapter 3 we examine the role of the adaptor protein BICD2 in the development of the cerebellum. This protein connects the motor protein dynein with the cargo vesicle. Removing this protein in mice leads to a completely disrupted cerebellar development, where the neurons do not migrate to their proper place in the cerebellar cortex. By removing the protein exclusively in one specific type of cells, we have shown that this developmental disorder only occurs when BICD2 is no longer present in Bergmann glia cells. In this celltype-specific knock-out, we found a reduction of adhesion molecules in the cellular membrane, possibly leading to a disrupted contact between these glia cells and the neurons migrating along them.

Chapter 4 provides an overview of literature on the role of intracellular transport in MS and presents a model to explain the contribution of transport to the development of this disease. In chapter 5 we show the result of inducing experimental autoimmune encephalomyelitis (EAE) in a mouse in which one of the genes encoding the molecular motor KIF1B has been knocked out. Knocking down both copies of this gene is not compatible with life, but we found that removal of just one copy does not influence susceptibility to or severity of EAE. Since EAE was primarily designed as a model to study the immunological aspects of EAE, in chapter 6 we present a number of analysis methods designed to increase the value of this model in studying MS. Research on intracellular transport is highly dependent on advanced microscopy meth- ods to visualise the movement of motor proteins and cargo. In chapter 7 we explain how light microscopy is limited in resolution because of the physical properties of light and how this limit can be broken. The background of superresolution microscopy is explained, as well as methods to better calculate the achieved resolution and further improve the image quality through drift correction and labelling techniques. In chap- ter 8 we apply these techniques by labelling the microtubule cytoskeleton with a VHH antibody fragment targetting tubulin. Finally chapter 9 summarizes the findings of this thesis and proposes a number of recommendations for future research.