The tight spatial coupling of synaptic vesicles and voltage-gated Ca 2+ channels (Ca V s) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca 2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased shortterm facilitation. Superresolution microscopy revealed an impairment in Ca V 2.1 clustering at AZs, which likely alters Ca 2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.RIM-BP2 | calcium channel coupling | release probability | short-term plasticity | active zone structure A t the presynapse, coupling between action potentials (APs) and synaptic vesicle fusion is exquisitely precise, ensuring high temporal fidelity of neuron-to-neuron signaling in the nervous system. Two properties are thought to be responsible for this remarkable precision: a highly efficient release apparatus that transduces Ca 2+ signals into vesicle fusion and a tightly organized active zone (AZ), where the release apparatus and voltage-gated Ca 2+ channels (Ca V s) are spatially coupled. Rab-interacting molecules (RIM) are thought to contribute to both properties, because loss of RIM impairs vesicle priming (1) and Ca V localization at the AZ (2). RIM-binding proteins (RIM-BPs) directly interact with RIM (3), the pore-forming subunits of Ca V 1 and Ca V 2 channels (2, 4, 5), and Bassoon (5), and have therefore been suggested to play a role in presynaptic Ca V localization. The Drosophila homolog of RIM-binding proteins (DRBP) is indeed crucial for neurotransmitter release at the AZ of neuromuscular junctions (NMJs) because loss of DRBP reduces Ca V abundance and impairs the integrity of the AZ scaffold (6). DRBP-deficient flies show severe impairment of neurotransmitter release along with increased short-term facilitation (6, 7).Recently, Acuna et al. (8) published a report on the combined loss of RIM-BP1 and RIM-BP2 in mouse synapses. The authors report that although RIM-BPs are not essential for synaptic transmission, AP-triggered neurotransmitter release is more variable and the sensitivity to the Ca 2+ chelator EGTA is increased at the Calyx of Held, suggesting a larger coupling distance of Ca V and the release machinery.In the present study, we further investigated the consequences of constitutive deletion of RIM-BP2 on the structure and function of mouse hippocampal synapses. We show that loss of RIM-BP2 lead...

All synapses require fusion-competent vesicles and coordinated Ca2+-secretion coupling for neurotransmission, yet functional and anatomical properties are diverse across different synapse types. We show that the presynaptic protein RIM-BP2 has diversified functions in neurotransmitter release at different central murine synapses and thus contributes to synaptic diversity. At hippocampal pyramidal CA3-CA1 synapses, RIM-BP2 loss has a mild effect on neurotransmitter release, by only regulating Ca2+-secretion coupling. However, at hippocampal mossy fiber synapses, RIM-BP2 has a substantial impact on neurotransmitter release by promoting vesicle docking/priming and vesicular release probability via stabilization of Munc13-1 at the active zone. We suggest that differences in the active zone organization may dictate the role a protein plays in synaptic transmission and that differences in active zone architecture is a major determinant factor in the functional diversity of synapses.

1All synapses require fusion--competent vesicles and coordinated Ca 2+ --secretion coupling for 2 neurotransmission, yet functional and anatomical properties show a high diversity across different 3 synapse types. We show here that the presynaptic protein RIM--BP2 has diversified functions in 4 neurotransmitter release at different central mammalian synapses and thus contributes to 5 synaptic diversity. At hippocampal pyramidal CA3--CA1 synapses, RIM--BP2 loss has a mild effect on 6 neurotransmitter release, by only regulating Ca 2+ --secretion coupling. However, at hippocampal 7 mossy fiber synapses RIM--BP2 has a strong impact on neurotransmitter release by promoting 8 vesicle docking/priming via recruitment of Munc13--1. In wild type mossy fiber synapses, the 9 distance between RIM--BP2 clusters and Munc13--1 clusters is larger than in hippocampal pyramidal 10 CA3--CA1 synapses, suggesting that spatial organization may dictate the role a protein plays in 11 synaptic transmission and that differences in active zone architecture is a major determinant factor 12 in the functional diversity of synapses . 13 14 15 16 17 18

Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer’s disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions.

The dynamic regulation of the actin cytoskeleton plays a key role in controlling the structure and function of synapses. It is vital for activity-dependent modulation of synaptic transmission and long-term changes in synaptic morphology associated with memory consolidation. Several regulators of actin dynamics at the synapse have been identified, of which a salient one is the postsynaptic actin stabilising protein Drebrin (DBN). It has been suggested that DBN modulates neurotransmission and changes in dendritic spine morphology associated with synaptic plasticity. Given that a decrease in DBN levels is correlated with cognitive deficits associated with ageing and dementia, it was hypothesised that DBN protein abundance instructs the integrity and function of synapses. We created a novel DBN deficient mouse line. Analysis of gross brain and neuronal morphology revealed no phenotype in the absence of DBN. Electrophysiological recordings in acute hippocampal slices and primary hippocampal neuronal cultures showed that basal synaptic transmission, and both long-term and homeostatic synaptic plasticity were unchanged, suggesting that loss of DBN is not sufficient in inducing synapse dysfunction. We propose that the overall lack of changes in synaptic function and plasticity in DBN deficient mice may indicate robust compensatory mechanisms that safeguard cytoskeleton dynamics at the synapse.