|M.Sc Student||Geva Ran|
|Subject||Maintenance of the Post Synaptic Density|
|Department||Department of Medicine||Supervisor||Professor Noam Ziv|
|Full Thesis text - in Hebrew|
Recent studies suggest that central nervous system (CNS) synapses persist for many weeks, months and even lifetimes, yet little is known on the mechanisms that allow these structures to persist for so long despite the many deconstructive processes acting at biological systems and neurons in particular. As a step toward a better understanding of synaptic maintenance we set out to examine some of the deconstructive and reconstructive forces acting at individual post synaptic densities (PSD) in CNS synapses. To that end we studied the molecular dynamics of the postsynaptic cytomatrix molecule, ProSAP2. Fluorescence recovery after photobleaching (FRAP) experiments of GFP-tagged ProSAP2 revealed that these molecules are continuously incorporated into and lost from individual synaptic structures within tens of minutes. Moreover, these dynamics are greatly accelerated by synaptic activity. To study the trafficking of ProSAP2 within dendrites we exchanged the GFP moiety with photoactivatable (PA)-GFP. Before photoactivation PA-GFP:ProSAP2 was not readily resolved, while after photoactivation it was easily detected. This technique enabled us to measure the residence time of ProSAP2 at individual dendritic spines, and to determine that ProSAP2 lost from individual PSDs is incorporated rapidly (tens of minutes) into the PSDs of neighboring spines. Conversely, these experiments showed that incorporation rates of ProSAP2 delivered from somatic sources are relatively slow (many hours). In order to clarify whether there is a measurable effect of protein synthesis and degradation on ProSAP2/Shank3 exchange dynamics we used the protein synthesis inhibitor cycloheximide and the proteasome-mediated protein degradation inhibitor MG132. Steady state levels or exchange rates of GFP:ProSAP2/Shank3 were not affected by cycloheximide. However, MG132 did increase steady state levels of GFP:ProSAP2/Shank3 in about half of neurons examined, but not in others. Neither inhibitor significantly affected exchange kinetics measured by FRAP within a time frame of about one hour.
The experiments described above strongly indicate that ProSAP2/Shank3 is continuously lost from, redistributed among, and reincorporated into postsynaptic structures at timescales of minutes to hours. These findings indicate that the dynamics of key PSD proteins, at least insofar as ProSAP2 is concerned, are dominated by local protein exchange and redistribution whereas protein synthesis and degradation seem to be second-order processes that serve to maintain and regulate the size of the local, shared synaptic protein pools.