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Presynaptic Regulatory Principles in Synaptic Plasticity, Neuronal Network Function, and Behaviour

Periodic Reporting for period 3 - SYNPRIME (Presynaptic Regulatory Principles in Synaptic Plasticity, Neuronal Network Function, and Behaviour)

Reporting period: 2018-09-01 to 2020-02-29

General Abstract
SynPrime focuses on the key signaling processes by which neurons communicate in the brain. Neuronal signaling takes place at synapses, where sending nerve cells release small molecules - neurotransmitters - to receiving cells. The speed of transmitter release and the ability of synapses to sustain it at high stimulation rates are key requirements for brain function, and plastic changes of transmitter release rates are thought to control multiple complex brain processes, such as working memory, sensory adaptation, or sound localization. Accordingly, many neuropsychiatric disorders involve dysfunctional synapses. We found previously that patients with mutations in Munc13-1, a 'master-controller' of transmitter release, suffer from dyskinesia and cognitive and behavioral defects, and showed that these mutations perturb the fine-tuning of synaptic signaling. During the previous and current reporting periods, we revealed key aspects of the molecular and cellular mechanisms by which Munc13 proteins render transmitter-filled secretory vesicles 'fusion-ready', so that synapses can respond to a stimulus by immediate transmitter release and recover quickly during and after phases of strong stimulation. Further work focused on the role of the Munc13-related protein CAPS. Here we found, using electrophysiological recordings from anesthetized mice, that CAPSs control the process of sensory adaptation in the visual system of the brain in vivo. In a third important project, using newly generated genetically modified mouse models, we studied the role of Complexins, which control the speed and efficacy of vesicle fusion during transmitter release and have been linked to multiple brain disorders, including schizophrenia. We found, in contrast to many published data, that Complexins are positive regulators of synaptic vesicle fusion. In two as yet unpublished studies, we used methods that allow us to arrest synapses in defined functional states to show that depressed synapses have less fusion-ready transmitter-filled, and we discovered that Munc13s are tightly controlled by calcium ions and membrane lipids in vivo, so that their activity is properly adjusted to synaptic demand. On aggregate, our work revealed important new key insights into the physiology and pathophysiology of neurotransmitter signaling in the brain.

Background
Nerve cell signaling via synaptic vesicle (SV) fusion is the fastest membrane fusion event in mammalian cells. Its speed and the ability of synapses 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, sensory adaptation, 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 'fusion-ready', primed SV pool that can be replenished rapidly. SV priming is mediated by 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 newly generated conditional knock-out (KO) and knock-in (KI) mouse lines, 17 additional KOs/KIs (12 ours), high-end electron microscopy approaches, KO-replacement strategies, electrophysiological and optophysiological analyses, and behavioral studies, we are examining in this project the SV priming machinery in intact circuits. Our aims are to identify the mechanisms and cell biological basis
During the first two SynPrime reporting periods, we made three major discoveries. First, we examined the role of synaptic activity in the formation and maintenance of synaptic connectivity in the brain, and found that - contrary to dogma - the formation and maintenance of neuronal morphology and synapses are strikingly independent of synaptic signalling, indicating that brain circuit connectivity is initially established by activity-independent cellular programs (Sigler et al., 2017). Second, we showed 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. This underscores the critical importance of fine-tuned presynaptic control in normal brain function and defines Munc13s and SV priming as new etiological factors in neuropsychiatric synaptopathies (Lipstein et al., 2017). Third, we showed that synapse-specific interactions of different Munc13s with ELKS1 or RIMs determine the molecular and functional heterogeneity of presynaptic AZs (Kawabe et al., 2017). This finding makes an important contribution to our understanding of how functional synapse diversity is generated in the brain.

During the current reporting period, we revealed further key aspects of the molecular and cellular mechanisms by which Munc13 proteins render transmitter-filled secretory vesicles 'fusion-ready', so that synapses can respond to a stimulus by immediate transmitter release. Our data in this regard indicate that synaptic transmitter release probability and short-term plasticity are - at least in part - dictated by the number of membrane-attached SVs and the ratio between these membrane-attached SVs and membrane-proximal SVs (Maus et al., 2020). Second, we studied the Munc13-related protein CAPS and found, using electrophysiological recordings from anesthetized mice, that CAPS-dependent SV priming controls the process of adaptation at thalamo-cortical synapses in the visual system of the brain in vivo (Nestvogel et al., 2020). This represents the first direct evidence of a role of SV priming in a complex computational process in the brain. In a third important project, using newly generated genetically modified mouse models, we studied the role of Complexins, which control the speed and efficacy of vesicle fusion during transmitter release and have been linked to multiple brain disorders, such as schizophrenia. Our data show - in contrast to many published data - that Complexins are positive regulators of synaptic vesicle fusion and do not act as fusion clamps in mammalian synapses (Lopez-Murcia et al., 2020).

In two as yet unpublished studies, which are now ready for submission, we used optogenetic and high-pressure freezing methods that allow us to arrest synapses in defined functional states (e.g. strengthened or weakened) to show that the number of fusion-ready transmitter-filled SVs is reduced in weakened, short-term depressed synapses, and we discovered that Munc13 proteins are tightly controlled by calcium ions and membrane lipids. As regards the first of these studies, our data show that synaptic depression in hippocampal synapses is, at least in part, caused by a partial depletion of membrane-attached SVs. As regards the second study, we examined newly generated knock-in mutant mice expressing mutant Munc13-1 variants with 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.

On aggregate, our work revealed important new insights into the physiology and pathophysiology of synapses in the brain.
Of the work published so far, six 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. 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 20 other patients with Munc13-1 mutations, which we are currently studying. Some of these patients have the same de-novo mutation as the one we initially characterized, indicating a mutational hot-spot. 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 the role of CAPSs in controlling presynaptic short-term plasticity at thalamo-cortical synapses in vivo is the first-ever indication that SV priming and the corresponding short-term plasticity characteristics may indeed shape sensory adaptation, as has long been hypothesized (Nestvogel et al., 2020). Fourth, our work on a newly generated conditional Complexin-1 knock-out mouse line provided a clarification of a very contentious issue in the field, showing that Complexins act as positive regulators of SV fusion and not as fusion clamps in mammalian synapses (Lopez-Murcia et al., 2020). Fifth, our work combining optogenetic stimulation with high-pressure freezing and electron microscopic analysis using hippocampal organotypic slices allowed us to define the morphological manifestation of presynaptic depression, i.e. a partial depletion of membrane-attached, primed SVs. These findings are paralleled by a recent study of ours showing that synaptic transmitter release probability and short-term plasticity are, at least in part, dictated by the number of membrane-attached SVs and the ratio between these and membrane-proximal SVs (Maus et al., 2020). Sixth, our analysis of the regulation of Munc13-1 by second messengers shows for the first time in intact circuits how massive the influence of SV priming proteins on synapse features really is.

At this point, the 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.