RESOLVING MILLISECOND CHANGES TO SYNAPTIC PHOSPHOSITES IN RESPONSE TO NEURONAL ACTIVITY

Synaptic vesicle fusion at the presynaptic active zone is orchestrated by a large number of proteins that make up the release machinery. Many of these proteins are substrates of kinases and phosphatases, and their function is dynamically regulated by phosphorylation. Neuronal activity regulates the activity of many kinases and phosphatases and thereby, the synaptic phosphorylation landscape. However, it is unknown how many action potentials are required to induce synaptic phospho-patterns, how quickly phosphorylation builds up, and how persistent it is. This information is key to understanding the role of synaptic phosphorylation during physiological patterns of neuronal activity. Therefore, we have established a ms-delay optogenetic-biochemical pipeline in which samples are flash-frozen in liquid nitrogen immediately after patterned optogenetic stimulation, to capture phospho-states at defined time points. We evoked neurotransmitter release with the channelrhodopsin CheRiff and quantified phosphorylation changes using targeted phospho-immunoblotting and unbiased phospho-proteomics. Across synaptic proteins, we observed specific positive or negative phosphorylation patterns which were sensitive to frequency and duration of stimulation. Our results show that already a short train of action potentials (such as 100 ms, 20Hz) triggers changes in phosphosites (like Synapsin I serine-9) indicating that the presynaptic phosphoproteome is a rapid integrator of presynaptic activity and a prime candidate of a biochemical correlate of the functional state of a synapse and also an important part of quick storage of information in the brain.