Ph.D Thesis

Ph.D StudentTsuriel Shlomo
SubjectCellular and Molecular Dynamics Involved in The Maintenance
of Presynaptic Structure
DepartmentDepartment of Medicine
Supervisor PROF. Noam Ziv
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Much is known about the structure, molecular composition and physiology of CNS synapses. Conversely, little is known about the mechanisms that maintain the structural integrity and functional characteristics of synapses over time. This is particularly true for presynaptic compartments, specialized axonal domains continuous with the axon but not confined by physical borders. Here we evaluated the challenges faced by synaptic maintenance processes and exposed principles of synaptic maintenance by studying the exchange, redistribution and replenishment dynamics of four representative presynaptic molecules. To that end we used fluorescently-tagged presynaptic proteins, advanced imaging methodologies and primary cultures of rat hippocampal neurons.

Experiments performed with the cytomatrix protein Synapsin I revealed that this protein is continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of tens of minutes. These rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation. In contrast, replenishment from somatic sources was much slower.

Similar exchange and redistribution dynamics were observed for two additional presynaptic proteins. The v-SNARE protein VAMP2 exhibited short residency times at synapses (~minutes), whereas the membranal tSNARE protein Syntaxin1A, exhibited longer residency times, on the order of ~2 hours. Both proteins appeared to be transported from the cell body by fast axonal transport but incorporation rates of proteins from somatic scores was much slower than exchange rates measured at individual boutons.

Finally, the dynamics of the active-zone molecule Bassoon, were examined with the assumption that this huge protein may be part of a relatively stable core scaffold, that nucleates and maintains presynaptic organization. Our experiments revealed that the exchange rates of GFP-tagged Bassoon at individual presynaptic sites are very low (τ≈4hr) and varied greatly across the population. Furthermore, a significant fraction of synapses exhibited much slower or undetectable exchange rates. Unlike other synaptic proteins we examined, electrical stimulation had only minor effects on Bassoon exchange rates. These findings indicate that Bassoon is engaged in relatively stable associations, and thus may be part of a relatively stable presynaptic core scaffold.

In summary, our findings indicate that the dynamics of presynaptic molecules are dominated by local protein exchange and redistribution whereas protein synthesis and degradation mainly serve to maintain and regulate the sizes of local, shared pools of these proteins. Our findings also support the possibility that the location and particular characteristics of individual presynaptic sites is maintained and dictated by relatively stable core scaffolds that reside in the active zone.