Cytoskeletal mechanisms of synaptic cargo trafficking

The brain is a complex organ, responsible for the most important tasks performed by humans and animals. Neurons are an extraordinary type of brain cells. Their particular shape, with dendrites and axons that extend over long distances, allows one neuron to communicate with a neighboring neuron or with neurons that are far apart. Communication between neurons occurs through synapses. To ensure faithful communication between neurons, synapses must be correctly located and assembled. Trafficking of cargo to synapses is a complex event, which is currently not completely understood. The cytoskeleton provides the roads for motor proteins to walk and deliver synaptic cargo to its final destination. How the cytoskeleton is organized along the neuron, what activates or inhibits motor proteins to transport cargo and which cargo is targeted for transport and where is this determined are questions not completely answered yet. With the use of different approaches and methodologies this thesis describes our effort in understanding synaptic cargo trafficking in hippocampal neurons. Chapters 2 and 3 focus on the postsynapse. In hippocampal neurons, most excitatory synapses are located in dendritic spines. At the surface of the postsynapse, the signal from the presynaptic axon is received by membrane receptors, such as AMPA receptors. In chapter 2 we describe the trafficking of AMPA receptors to dendritic spines inside recycling endosomes via the microtubule and the actin cytoskeleton, and how it is possible to control the insertion or removal of AMPA receptors from the postsynapse with consequences to the organization and functionality of the postsynapse. In chapter 3, our studies in the postsynapse focused on microtubule invasions of dendritic spines. Microtubules are typically located along the dendritic shaft and microtubule entries in spines are not a frequent and well understood event. These events were studied in great detail, as well as its functional implications in basal conditions or upon synaptic plasticity. Motor proteins are fundamental for transport inside cells, including correct delivering of cargo to the synapse. In chapter 4, the regulation of the dendritic kinesin KIF17 was studied, and by coupling KIF17 to specific cargo we provided novel insights on how this motor protein is distinctly regulated by its own tail domain and by binding to cargo. In chapter 5, the axonal actin cytoskeleton and its role in presynaptic cargo trafficking was explored. This chapter studies the distribution and organization of actin structures along the axonal shaft. The results presented in this chapter indicate that a family of actin nucleators are important in the targeting of cargo to the presynapse. Finally, chapter 6 describes the development and characterization of novel tools to manipulate the actin cytoskeleton. DeActs were studied in different model systems, highlighting their robustness and wide applicability. Ì believe that DeActs will be a widely used approach in different experimental setups and will help to better understand the role of actin dynamics in cells. The results described in this thesis advance our knowledge of synaptic cargo trafficking and I am hopeful they will open doors for further studies in this field