Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SYNPRIME (Presynaptic Regulatory Principles in Synaptic Plasticity, Neuronal Network Function, and Behaviour)

Teaser

General AbstractThe SynPrime project is concerned with the signaling processes by which neurons communicate in the brain, and with the role that these processes play in the healthy and diseased brain. Inter-neuronal signaling occurs at so-called synapses, where a sending nerve...

Summary

General Abstract
The SynPrime project is concerned with the signaling processes by which neurons communicate in the brain, and with the role that these processes play in the healthy and diseased brain. Inter-neuronal signaling occurs at so-called synapses, where a sending nerve cell releases messenger substances - so-called neurotransmitters - to a receiving cell. The speed of this transmitter release process, and the ability of synapses to sustain transmitter release at high stimulation rates are key requirements for brain function. Indeed, plastic changes of synaptic transmitter release rates have long been thought to control complex brain processes such as working memory, sound localization, or gain control - and multiple psychiatric and neurological disorders are caused by dysfunctional synapses. In one SynPrime sub-project, we identified a mutation in the synaptic protein Munc13-1 in a patient with autism, hyperactivity, and dyskinesia, and found that this mutation perturbs the fine-tuning of synaptic signaling. Although the initially identified mutation itself is rare, we have now obtained information on at least five more rare, disease-related Munc13-1 mutations, which further stresses the notion that variations in the function of synaptic proteins may cause the symptoms seen in patients with ASD and other neuropsychiatric disorders. In addition, our work shows that Munc13 proteins may be potential targets for pharmacological intervention in autism and other neuropsychiatric disorders. In a second important SynPrime sub-project, we focused on the mechanisms by which synapses are generated in the first place. For a long time, it has been assumed that active transmitter release from nerve cells is required for the formation of so-called spine synapses. Spines are major neurotransmitter reception compartments of nerve cells. Contrary to the currently held dogma, we discovered that spines are formed independently of active inter-neuronal signaling, indicating that brain circuit connectivity is initially established by activity-independent cellular programs. In a third SynPrime project complex, we have characterized mechanisms by which the function of the key synaptic protein Munc13-1 is regulated by cellular signaling pathways in order to adapt synapse function to constantly changing demands. Our data in this regard show that Munc13s are key determinants of synapse endurance and fidelity in intact neuronal circuits. Finally, we have established methods that allow us to arrest synapses in defined functional states (e.g. strengthened or weakened) and to study their structure at the ultrastructural level using electron microscopy.

Background
Nerve cell signaling via synaptic vesicle (SV) fusion is the fastest membrane fusion event in mammalian cells. Its speed and the ability of presynapses to sustain SV fusion at high stimulation rates are key requirements for brain function. Indeed, plastic changes of SV fusion rates have long been thought to control complex brain processes such as working memory, sound localization, or gain control. However, the link between presynaptic plasticity and complex brain functions has remained hypothetical. A key determinant of presynaptic efficacy is that synapses maintain a release-ready or primed SV pool that can be replenished rapidly. SV priming is mediated by a set of dedicated priming proteins (Munc13s, CAPSs, and accessory proteins), which are of pervasive and essential functional importance for synaptic efficacy, and - based on in vitro studies - of capacious potential to regulate exactly the type of synaptic plasticity that is associated with brain circuit characteristics involved in complex behaviors. However, this \'catholic\' role of the SV priming machinery in brain function has never been tested, mainly because essential genetic models for studies in vivo have been lacking.

Overall Objectives
Based on (i) newly generated conditional knock-out (KO) and knock-in (KI) mouse lines, (i

Work performed

Towards the end of the first reporting period and in the beginning of the second, we completed and published three major SynPrime sub-projects. In one, which was published in the beginning of the second reporting period, we examined the role of synaptic activity in the formation and maintenance of synaptic connectivity in the brain. Our data show that - contrary to dogma - the formation and maintenance of neuronal morphology and of synapses are strikingly independent of synaptic signaling, indicating that brain circuit connectivity is initially established by activity-independent cellular programs (Sigler et al., 2017). A second study highlights the importance of Munc13-family priming proteins in proper brain function, showing that a rare, de novo Pro814Leu variant in the major human Munc13 paralog Munc13-1 causes dyskinetic movement disorder, developmental delay, and autism by increasing Munc13-1 function, boosting SV fusion propensity of SVs and transmitter release probability, and altering short-term synaptic plasticity. This study underscores the critical importance of fine-tuned presynaptic control in normal brain function and adds the neuronal Munc13 proteins and the SV priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies (Lipstein et al., 2017). In a third mayor SynPrime sub-project, we showed that synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs (Kawabe et al., 2017).

Beyond these published studies, we have invested all SynPrime resources in the second reporting period to further develop high-end electron microscopy and electrophysiological/optogenetic methods to study synapse function. Most importantly, we have now successfully established methods to optically and electrically stimulate synapses in brain slices, followed by high-pressure freezing and electron microscopic analyses. We are now using this methodology to determine the structural manifestations of defined synapse states at the electron microscopic level. In addition, we have almost completed the analysis of knock-in mutant mice that express mutant Munc13-1 variants that are characterized by either increased or decreased sensitivity to calcium and membrane phospholipids. Our corresponding data show that the regulation of the SV priming protein Munc13-1 by calcium and phospholipids - acting upon the so-called C2B domain - is a major determinant of synaptic endurance, fidelity, and plasticity in brain circuits as they occur in vivo.

Final results

Of the work published so far, four highlights represent substantial steps beyond the current state of the art. First, the new disease related Munc13-1 mutation we analyzed (Lipstein et al., 2017) was the first-ever disease-related Munc13-1 mutation discovered so far. As stated above, the corresponding study underscores the critical importance of fine-tuned presynaptic control in normal brain function and adds the neuronal Munc13 proteins and the SV priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies. Since the publication of our study, we have become aware of at least five other patients with Munc13-1 mutations, which we are currently studying. Second, our work demonstrating that spine generation and maintenance are independent of presynaptic glutamate release disproves the long-held dogma that spinogenesis requires glutamate release (Sigler et al., 2017). It is very likely that this study will lead to a general reassessment of the role of synaptic activity in circuit formation. Third, our work on combining optogenetic/electric stimulation with high-pressure freezing and electron microscopic analysis using hippocampal organotypic slices represents a major step forward in the cell biological analysis of synapse function. Finally, our analysis of the regulation of Munc13-1 by second messengers at the level of intact circuits shows for the first time in intact circuits how massive the influence of SV priming proteins on synapse features really is.

At this point, a possible socio-economic impact of our work is difficult to extrapolate. However, the ongoing analysis of patients with Munc13-1 mutations (Lipstein et al., 2017) might ultimately lead to therapeutic solutions. The increased synaptic release probability that characterizes the first patient we analyzed is - in principle - treatable with available drugs on the market. Beyond this, we trust that the other disease-related Munc13-1 mutations that have been discovered in the meantime - and possibly also CAPS mutations - will inform on relevant disease mechanisms and therapy strategies, so that our \'ground-work\' on the SV priming process might ultimately become clinically relevant.