Skip to main content

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

Periodic Reporting for period 4 - SYNPRIME (Presynaptic Regulatory Principles in Synaptic Plasticity, Neuronal Network Function, and Behaviour)

Reporting period: 2020-03-01 to 2020-08-31

General Abstract
SynPrime focuses on neuronal signalling in the brain, which occurs at synapses where sending cells release neurotransmitters from synaptic vesicles (SVs) that fuse with the plasma membrane. The speed of SV fusion and the ability of synapses to sustain it at high rates are key requirements for brain function, and dynamic changes of the process are thought to control complex brain processes. Accordingly, many brain disorders involve dysfunctional synapses. We found that patients with mutations in Munc13-1, a 'master-controller' of SVs, suffer from dyskinetic, cognitive, and behavioral defects, and showed that these mutations perturb synapse fine-tuning. We revealed the mechanisms by which Munc13 proteins render SVs 'fusion-ready', so that stimulated synapses respond by immediate SV fusion and recover quickly after phases of strong stimulation. Work on Munc13-related CAPS proteins showed that they control sensory adaptation in the visual system of the brain in vivo. Using newly generated mutant mice, we studied Complexins, which control the efficacy of SV fusion and are linked to multiple brain disorders. We found, in contrast to dogma, that they are positive regulators of SV fusion and not fusion clamps. Using novel methods to characterize synapses in defined functional states, we showed that depressed synapses have less fusion-ready SVs and discovered that Munc13s are tightly controlled by calcium and membrane lipids in vivo, so that their activity is adjusted to synaptic demand. Our work revealed key insights into the physiology and pathophysiology of neurotransmitter signaling.

The speed of SV fusion and the ability of synapses to sustain it at high rates are key requirements for brain function. Plastic changes in these processes have long been thought to control complex brain processes, from sensory adaptation to working memory, but the link between presynaptic plasticity and complex brain functions has long 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 proteins (Munc13s, CAPSs, accessory proteins such as Complexins), which are essential for synaptic efficacy and of capacious potential to regulate synaptic plasticity associated with circuit characteristics that control behavior. However, this 'catholic' role of the SV priming machinery has long remained unproven in intact circuits.

Overall Objectives
Our aim was to examine the SV priming machinery in intact circuits and in vivo - specifically to identify the mechanisms of SV priming, of its dynamics, and of defined priming-dependent synaptic plasticity states, and to define the causal links between SV priming, synapse function, synaptic plasticity, and circuit characteristics that determine behavior.

Our project made a substantial contribution to a comprehensive description of the role of SV priming in intact circuits. This is important for basic and translational science alike, because all key SV priming proteins are linked to neuropsychiatric diseases.
(i) We showed contrary to dogma that the formation and maintenance of neuronal morphology and synapses are independent of synaptic signalling, indicating that brain circuit connectivity is initially established by activity-independent cellular programs (Neuron 2017).

(ii) We showed that a mutant human Munc13-1 variant causes a complex neuropsychiatric condition by increasing Munc13-1 function, underscoring the importance of presynaptic control in brain function and implicating Munc13s in the etiology of neuropsychiatric synaptopathies (Journal of Clinical Investigation 2017).

(iii) We showed that Munc13 interactions with ELKS1 or RIMs determine the functional heterogeneity of synapses, illustrating a mechanism by which synapse diversity in the brain is generated (J Cell Biol 2017).

(iv) We showed that synaptic transmitter release probability and short-term plasticity are dictated by the number of membrane-attached SVs and the ratio between these and membrane-proximal SVs (Cell Rep 2020).

(v) We showed that CAPS-dependent SV priming controls adaptation at thalamo-cortical synapses in the visual system in vivo, the first direct evidence of a role of SV priming in complex brain computation (Cell Rep 2020).

(vi) We showed contrary to dogma that Complexins, which control efficacy of SV fusion and are linked to multiple brain disorders, are positive regulators of SV fusion and not fusion clamps (Cell Rep 2020).

(vii) We developed novel technology to arrest synapses in defined functional states in intact tissue to show that synaptic depression is caused by partial depletion of membrane-attached SVs and that different endocytosis modes operate when synapses are stimulated extensively (Neuron 2020).

(viii) Using novel mutant mice, we showed that Munc13-1 regulation by calcium and phospholipids is a major determinant of synaptic endurance, fidelity, and plasticity in intact circuits.
(i) We identified the first disease-related Munc13-1 mutation (J Clin Invest 2017). Our study underscores the importance of presynaptic control in brain function and implicates Munc13s and SV priming in the etiology of synaptopathies. We have become aware of >30 other patients with Munc13-1 mutations, some of which have the same de-novo mutation as the one we characterized, indicating a mutational hot-spot.

(ii) Our demonstration that spine formation is independent of presynaptic glutamate release disproves a long-held dogma and will lead to a general reassessment of the role of synaptic activity in circuit formation (Neuron 2017).

(iii) Our data on CAPS function in presynaptic short-term plasticity at thalamo-cortical synapses in vivo provide the first support for the long-held hypothesis that SV priming and related short-term-plasticity processes shape sensory adaptation (Cell Reports 2020).

(iv) Our work on Complexin-1 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 (Cell Reports 2020).

(v) Our work combining optogenetic stimulation with high-pressure freezing and electron microscopy on hippocampal organotypic slices allowed us to define the morphological manifestation of presynaptic depression and identified key synaptic endocytosis steps. The new method will reshape ultrastructural analyses of synapses 'in action' (Neuron 2020).

The socio-economic impact of our work is difficult to extrapolate. Ongoing analyses of patients with Munc13-1 mutations might ultimately lead to therapeutic solutions. Beyond this, the other disease-related Munc13-1 mutations that have been discovered will inform on disease mechanisms and therapy strategies, so that our 'ground-work' might ultimately become clinically relevant.