Aim: 

In neurons, synaptic activity triggers the fusion of synaptic vesicles with the plasma membrane to release neurotransmitters. Endocytosis is required for neurotransmission since it reconstitutes the synaptic vesicle pool during sustained stimulation. A major pathway of internalization involves clathrin-mediated endocytosis, a process that requires a cascade of protein-protein and lipid-protein interactions. Components of this cascade include, besides clathrin and the clathrin adaptors, BAR-domain containing proteins, the GTPase dynamin and the phosphoinositide phosphatase synaptojanin. BAR (Bin-Amphiphysin-Rvs) domain containing proteins interact with the lipid bilayer to sense and propagate membrane curvature (through the BAR domain), and with dynamin, synaptojanin and numerous endocytic proteins (through the Src homology 3, SH3, domain). To gain insights into the precise function of some of these endocytic accessory factors in mammals, we use a reverse genetic approach in mice.

Methods: 

Homologous recombination was used to disrupt genes encoding accessory factors of clathrin-mediated endocytosis in mouse. The effect of genetic perturbation on the expression of various synaptic proteins has been analyzed. The consequence on synaptic structure and function was investigated by immunofluorescence and electron microscopy analysis.

Results: 

Ultrastructural analysis of mutant synapses revealed abnormalities in synaptic vesicle recycling, such as depletion of synaptic vesicles and a dramatic accumulation of clathrin-coated pits and/or vesicles surrounded by a dense cytomatrix. These changes correlate with structural abnormalities of axons detectable by immunofluorescence. Lipid assays and electrophysiological experiments are in progress to determine the impact of genetic perturbation on neuronal phosphoinositide turnover and on synaptic vescicle recycling, respectively.

Conclusion: 

Our data provides new mechanistic insight into the role of accessory factors in synaptic vesicle recycling, a process which is essential to sustain prolonged synaptic activity.