טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentCohen Laura
SubjectThe Role of Catabolic Pathways in Maintaining Synaptic
Proteostasis
DepartmentDepartment of Medicine
Supervisor Professor Noam Ziv


Abstract

Chemical synapses are sites of cell‐cell contact specialized for transmitting signals between neurons and other neurons, muscles or glands. Synapses are composed of neuronal membranes, synaptic vesicles and multiple integral, peripheral and interacting proteins, some of which play direct roles in synaptic transmission, whereas others regulate synaptic function or serve as structural scaffolds. As in nearly all biological structures, proteins at synapses have finite lifetimes. Thus, maintaining synaptic structure, function and specific characteristics depends on the continuous and precise provision of newly synthesized proteins and concomitant degradation of old and damaged protein copies.

While much has been learned on synaptic protein synthesis, little is known on the manners by which synaptic proteins are degraded. In Chapter 1, we describe experiments examining the involvement of the ubiquitin‐proteasomal system (UPS) in neuronal and synaptic protein degradation. Specifically, we examined how inhibiting this catabolic route affects the degradation rates of thousands of neuronal and synaptic proteins. In the course of this study we identified a number of proteins, including several proteins related to glutamate receptor trafficking, whose degradation rates were significantly slowed by UPS inhibition. Unexpectedly, however, degradation rates of most synaptic proteins were not significantly affected. Many of the differential effects of UPS inhibition were readily explained by a quantitative framework that considered known metabolic turnover rates for the same proteins. In contrast to the limited effects on protein degradation, UPS inhibition profoundly and preferentially suppressed the synthesis of a large number of synaptic proteins. These findings highlighted the importance of the UPS in the degradation of certain synaptic proteins, yet also indicated that under basal conditions most synaptic proteins seem to be degraded through alternative pathways.

In Chapter 2, we describe experiments aimed at uncovering aspects of neuronal structure and function that are most strongly dependent on the supply of newly synthesized protein copies, with the intention in mind to characterize the failure timeline when protein replenishment is suppressed. We found that suppressing protein synthesis for 24 hours using protein synthesis inhibitors is associated with modest synapse elimination, minor protein loss, mostly intact vesicle recycling, but enhanced erosion of synaptic configurations. Strikingly, pharmacological suppression of protein synthesis led to rapid (hours) reductions in spontaneous activity levels, as measured using multi‐electrode array substrates and extracellular recordings, pointing to the existence of functionally important proteins with short half-lives of unknown identity. Proteomic experiments revealed potential candidates, including protease inhibitors and calcium binding proteins.

In Chapter 3, we describe technologies we developed to study protein synthesis and trafficking in live neurons and quantify the effects of protein synthesis inhibitors on protein synthesis rates: In Section 3A, we describe HaloTag based constructs and the development of a non‐fluorescent HaloTag blocker for improved measurement and visualization of protein synthesis in living cells.

In Section 3B, we describe genetic code expansion as a tool to visualize synaptic protein synthesis in living neurons.