|M.Sc Student||Cohen Laura|
|Subject||Protein Turnover and Replenishment at Central Nervous System|
|Department||Department of Medicine||Supervisor||Professor Noam Ziv|
|Full Thesis text|
Synapses are cellular specializations by which neurons communicate among themselves and other cell types. Synapses are composed of proteins with finite lifetimes which need to be continuously replaced. Typical neurons form thousands of synapses, often at enormous distances from cellular biosynthetic centers and thus synaptic maintenance places significant metabolic and logistic demands on neurons. To date, the metabolic turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus the metabolic demands of synaptic proteostasis, the effects of distance from global protein synthesis sites or the availability of local protein synthesis facilities remain largely unknown.
Here we used primary cultures of rat cortical neurons, dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), quantitative immunohistochemistry and bioinformatics to measure the metabolic half-lives of hundreds of synaptic proteins (as well as thousands of neuronal proteins), examine how these depend on pre-/postsynaptic affiliation, association with particular molecular complexes, and on network activity levels. Most synaptic proteins identified here exhibited half-lifetimes of 2-5 days. Somewhat surprisingly, turnover rates were not significantly different for presynaptic or postsynaptic proteins, or for proteins whose mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited similar turnover rates, indicating that their biogenesis and degradation might be coupled. Collectively our findings suggest that ~0.7% of synaptic protein content is turned over every hour. Interestingly, suppressing network activity levels for many days resulted only in slight reductions of synaptic and neuronal protein half-lifetimes. Pharmacological suppression of protein synthesis, however, was followed by rapid (hours) reductions in spontaneous activity levels measured by means of multi-electrode arrays, pointing to the existence of functionally important proteins with much shorter half-lives.
As dendritic protein synthesis is thought to play important roles in both synaptic proteostasis and plasticity, we also studied the cellular distribution, stability, and dynamics of dendritic ribosomes using a fluorescent variant of the ribosomal subunit L10a. We found that EGFP-L10a fluorescence was mostly localized to somata and proximal dendrites, with very low levels observed in distal dendrites. Fluorescence recovery after photobleaching experiments indicated that dendritic ribosomes were rather immobile, and that their mobility was unaffected by several stimulation paradigms.
This study provides a first systematic analysis of synaptic protein turnover kinetics and consequentially, places physiological constraints on proposed relationships between exchange, synthesis and degradation of synaptic proteins on the one hand, and synaptic maintenance, tenacity and plasticity on the other.