From the DEPARTMENT OF NEUROSCIENCE Karolinska Institutet, Stockholm, Sweden MOLECULAR MECHANISMS IN SYNAPTIC VESICLE RECYCYLING

Neurons communicate through specialized contacts called synapses, which consist of preand postsynaptic compartments separated by a narrow synaptic cleft. The stimulusdependent rapid influx of calcium ions into the presynaptic compartment induces fusion of neurotransmitter-filled vesicles with the plasma membrane and release of their content into the synaptic cleft. The vesicle membrane then needs to be recycled to avoid disruption of synapse morphology and depletion of synaptic vesicles. Three pathways have been described in the literature: clathrin-mediated endocytosis in which vesicles already the size of synaptic vesicles are retrieved at the periactive zone, bulk endocytosis in which larger pieces of membrane are retrieved, and kiss-and-run in which the vesicle remains intact and forms only a transient fusion pore through which neurotransmitter is released. In this thesis, the molecular mechanisms of synaptic vesicle recycling were studied using acute perturbations in lamprey giant reticulospinal axons. The aim was to clarify three different aspects in the recycling machinery: first – how the clathrin coat assembly is regulated, second – how dynamin-dependent fission is regulated, and third – how the synapse copes with intense activity. Three proteins (epsin, EHD, and syndapin) were cloned in Lampetra fluviatilis using PCR-based methods. Binding partners were identified in pull-down assays and lipid-interacting properties were investigated in liposome sedimentation, tubulation, and budding assays. The cloned proteins were further used in the immunization of rabbits for the generation of domain-specific antibodies. These antibodies were used for Western blot, immunoprecipitations, immunolocalization, and presynaptic microinjections. Microinjection experiments were mainly analyzed with electron microscopy to reveal morphological phenotypes. All three proteins cloned in this thesis showed a high degree of homology in protein and lipid interacting regions with their mammalian orthologs as well as similar biochemical properties. The proteins were all localized to the pre-synaptic compartment where they were shown to accumulate at specific regions during synaptic activity: epsin – to the coat of clathrin-coated pits, EHD – to neck regions of GTP S induced endocytic structures, and syndapin – to the peri-active zone (excluding clathrin-coated pits). Perturbation of epsin, EHD, and syndapin in living synapses impaired synaptic vesicle recycling, observed as a loss of synaptic vesicles. Disruption of epsin’s Nterminal region inhibited coat assembly while perturbation of epsin’s clathrin/AP-2binding domain resulted in abnormal coat assembly. Perturbation of EHD resulted in an accumulation of clathrin coated pits with long necks indicating an impairment of endocytic fission. The perturbation of syndapin was associated with a massive accumulation of membranous cisternae and invaginations, but not of clathrin-coated pits at the plasma membrane. The findings in this thesis suggest that epsin links membrane deformation to clathrin-coated pit formation with a potential role in regulating the size of synaptic vesicles, that EHD regulates dynamin-mediated fission in an ATP-dependent fashion, and that syndapin is required for bulk endocytosis and for stabilizing the plasma membrane during intense synaptic activity. Thus, this thesis has added new knowledge about the molecular network that mediates synaptic vesicle recycling. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to in the text according to their Roman numerals: