Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - SynDegrade (The Role of Local Protein Degradation in Neurotransmitter Release and Homeostatic Plasticity)
Teaser
Robust animal behavior presupposes stable neural function. Chemical synapses are the major sites of information transfer in the nervous system. While many synapses reliably transmit information for decades, the proteins determining synaptic transmission have half-lives of...
Summary
Robust animal behavior presupposes stable neural function. Chemical synapses are the major sites of information transfer in the nervous system. While many synapses reliably transmit information for decades, the proteins determining synaptic transmission have half-lives of hours to weeks. It is currently unclear how stable synaptic transmission, and thus animal behavior, can be achieved in the light of constant protein turnover.
Several neurological diseases, such as certain forms of epilepsy or migraine, have been linked to unstable neural function. Moreover, maladaptive protein turnover has been implicated in a number of neural pathologies.
The major goal of this project is to uncover links between the mechanisms controlling local protein turnover at synapses and the homeostatic stabilization of synaptic transmission. To this end, we focus on the role of a major protein degradation pathway – the Ubiquitin Proteasome System (UPS) – in regulating neurotransmitter release. We are in the process of systematically investigating UPS-dependent modulation of synaptic transmission after genetic perturbation of genes encoding UPS components and synaptic proteins. In a first step, forward genetics and detailed electrophysiological analysis of synaptic transmission are used to identify genes required for UPS-dependent regulation of release. This analysis is currently only feasible in Drosophila. In a next step, we translate our findings into the mammalian central nervous system (CNS) by studying synaptic transmission at a CNS synapse that allows for detailed analysis of presynaptic function.
Work performed
We realized ~2/3 of the electrophysiology-based genetic screen in Drosophila designed to unravel the roles of major synaptic genes in UPS-dependent regulation of neurotransmitter release. The screen has identified two genes previously linked to neurological disease in humans and homeostatic synaptic plasticity in Drosophila.
We completed an electrophysiology-based genetic screen in Drosophila targeting 185 genes encoding E3 ligases, core components of the UPS. This RNAi screen has uncovered a number of candidate genes with altered synaptic transmission. We are in the process of validating and further analysing these candidate genes.
We established the infrastructure to study synaptic transmission and presynaptic physiology in acute mouse brain slices in order to transfer the knowledge from Drosophila into the mammalian CNS. Electrophysiological recordings from synapses in the cerebellum revealed distinct changes in synaptic function after acute pharmacological proteasome perturbation. Based on the first evidence for rapid homeostatic regulation of neurotransmitter release after acute glutamate receptor inhibition in the mammalian CNS, we started investigating homeostatic plasticity after proteasome perturbation.
Final results
We have linked new genes to UPS-dependent regulation of neurotransmitter release and homeostatic plasticity. We expect to finalize the detailed analysis of these candidate genes until the end of the project.
We generated several transgenic Drosophila lines for acute, optogenetic protein perturbation. We also engineered flies expressing a modified genetically-encoded calcium indicator that undergoes photo-conversion upon calcium binding and light exposure to monitor synaptic transmission. We are in the process of validating these stocks and expect to complete the validation and to use these reagents to advance the other aims before the end of the project.
We translated the phenomenology of presynaptic homeostatic plasticity from Drosophila into the mammalian CNS. In addition, we established the first links between proteasome function and neurotransmitter release modulation in acute brain slices. We plan on studying the presynaptic physiology of UPS-dependent control of release through direct presynaptic whole-cell patch clamp recordings. We will complete the analysis of UPS-dependent regulation of release during presynaptic homeostatic plasticity and test the molecular mechanisms identified in Drosophila in the mammalian CNS.