Munc13 supports fusogenicity of non-docked vesicles at synapses with disrupted active zones

  1. Chao Tan
  2. Giovanni de Nola
  3. Claire Qiao
  4. Cordelia Imig
  5. Richard T Born
  6. Nils Brose
  7. Pascal S Kaeser

  • Curated by eLife

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    Tan and colleagues studied synaptic transmission, presynaptic protein levels, and synaptic ultra-structure in hippocampal cultures of mice lacking the key active-zone proteins RIM (1, 2), ELKS (1, 2), and Munc13 (1, 2). Compared to cultures lacking only RIM and ELKS, additional deletion of Munc13 results in a further decrease of synaptic Munc13-1 levels, a similar reduction of the number of docked synaptic vesicles, and a more pronounced decrease of total synaptic vesicle number. At the physiological level, these RIM-ELKS-Munc13 hextuple knockout cultures display a further decrease in the pool of release-ready synaptic vesicles with largely unchanged release probability compared with RIM-ELKS quadruple KO cultures. The results support the conclusion of the nonredundant role of Munc13 in synaptic vesicle priming. On the other hand, while the genetic removal of all six genes involved clearly require the use of conditional KO mice, the resulting outcome of the experimental design is a hypomorphic phenotype, as neurotransmitter release is still detected and this complicates the interpretation of the findings. Overall, this study reinforces the notion that synapse formation is a remarkably resilient process that occurs even under strong perturbation of presynaptic function.

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Abstract

Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to voltage-gated Ca 2+ channels, and direct neurotransmitter release toward postsynaptic receptor domains. Simultaneous RIM + ELKS ablation disrupts these scaffolds, abolishes vesicle docking, and removes active zone-targeted Munc13, but some vesicles remain releasable. To assess whether this enduring vesicular fusogenicity is mediated by non-active zone-anchored Munc13 or is Munc13-independent, we ablated Munc13-1 and Munc13-2 in addition to RIM + ELKS in mouse hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM + ELKS impaired action potential-evoked vesicle fusion more strongly than RIM + ELKS knockout by further decreasing the releasable vesicle pool. Hence, Munc13 can support some fusogenicity without RIM and ELKS, and presynaptic recruitment of Munc13, even without active zone anchoring, suffices to generate some fusion-competent vesicles.

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  5. eLife

    eLife assessment

    Tan and colleagues studied synaptic transmission, presynaptic protein levels, and synaptic ultra-structure in hippocampal cultures of mice lacking the key active-zone proteins RIM (1, 2), ELKS (1, 2), and Munc13 (1, 2). Compared to cultures lacking only RIM and ELKS, additional deletion of Munc13 results in a further decrease of synaptic Munc13-1 levels, a similar reduction of the number of docked synaptic vesicles, and a more pronounced decrease of total synaptic vesicle number. At the physiological level, these RIM-ELKS-Munc13 hextuple knockout cultures display a further decrease in the pool of release-ready synaptic vesicles with largely unchanged release probability compared with RIM-ELKS quadruple KO cultures. The results support the conclusion of the nonredundant role of Munc13 in synaptic vesicle priming. On the other hand, while the genetic removal of all six genes involved clearly require the use of conditional KO mice, the resulting outcome of the experimental design is a hypomorphic phenotype, as neurotransmitter release is still detected and this complicates the interpretation of the findings. Overall, this study reinforces the notion that synapse formation is a remarkably resilient process that occurs even under strong perturbation of presynaptic function.

  6. eLife

    Reviewer #1 (Public Review):

    Tan and colleagues examine the role of additional genetic removal of Munc13 in murine cultured synapses deficient for RIM and ELKS. They utilize a comprehensive set of morphological, ultrastructural and functional experiments to conclude that Munc13 is a nonredundent factor in synaptic vesicle priming. In addition, the results contribute to the ongoing discussion whether synaptic vesicle docking represents synaptic vesicle priming.

    The main strength of the work is the use of a sophisticated genetic model, and the quality of the performed experiments. The results strongly support the conclusion of the nonredundant role of Munc13 in synaptic vesicle priming.

    The weakness of the paper is that the findings from these study has limited impact, as the genetic and functional interaction of Munc13 and RIM has been extensively analyzed on a qualitative and quantitative level (Kaeser 2021, Zarebidaki 2020). While this paper benefits from the additional deletion of ELKS, the specific contribution of ELKS in comparison to the older studies is not in the focus of the study.

    While the genetic removal of all 6 genes involved clearly require the use of conditional KO mice, the resulting outcome of the experimental design is a hypomorphic phenotype, as neurotransmitter release is still detected. This complicates the interpretation of the findings and weakens the strength of the conclusions.

  7. eLife

    Reviewer #2 (Public Review):

    Tan et al. have used state-of-the-art methodology (mouse genetics, superresolution microscopy and synaptic electrophysiology) to further delineate the role of Munc13 proteins by investigating their function within a scenario in which the presynaptic active zone is deprived of major protein scaffolds. The authors have transduced Cre-expressing lentiviruses into hippocampal neuronal cultures from mice with floxed alleles to remove six fundamental components of the presynaptic active zone: RIM1, RIM2, ELKS1, ELKS2, Munc13-1, Munc13-2 and Munc13-3. The first part of the study comprises a comparison between neurons lacking RIM1, RIM2, ELKS1, ELKS2, on one side, and neurons lacking Munc13-1 and Munc13-2 on the other. Within the first group of neurons the levels of Munc13-1 at the nerve terminals are already reduced (assessed by confocal and STED microscopy and western blots) and the residual amount left is located far away from the active zone. Remarkably synapse formation occurs normally upon the hextuple knock-out of active zone proteins, however, vesicle docking is disrupted and single-action potential evoked and spontaneous release is reduced at glutamatergic and GABAergic synapses. A key finding is that the single-action potential evoked release still detected in the RIM/ELS cuadruple knock-out is almost completely abolished upon the additional knock-out of Munc13-1 and Munc13-2. This is a major observation of the study that support, as the authors concluded, that Munc13 promotes the fusogenicity of synaptic vesicles even when Munc13 is not properly located at the active zone. Careful electrophysiological measurements show that in the absence of Munc13 the size of the readily releasable pool (RRP) of synaptic vesicles is further reduced without specific changes in the vesicular release probability. Overall, the electrophysiological data support well the notion that Munc13 is specifically responsible for the remaining RRP and therefore reinforce the notion that Munc13 acts at the priming stage and can do it in part independently of RIMs and ELSs. Importantly, the results further support the notion that synapse formation is a remarkably resilient process that occurs even under strong perturbation of presynaptic function.

    As a secondary conclusion, the authors point out that postsynaptic response is intact, this specific point should be further discussed and analyzed.

    The study is very clearly written and presents very relevant findings of interest for readers in the field of the molecular mechanisms of synaptic operation.

  8. eLife

    Reviewer #3 (Public Review):

    In the present study, Tan and colleagues studied synaptic transmission, presynaptic protein levels, and synaptic ultra-structure in hippocampal cultures of mice lacking the key active-zone proteins RIM (1, 2), ELKS (1, 2), and Munc13 (1, 2). Compared to cultures lacking only RIM and ELKS, additional loss of Munc13 results in a further decrease of synaptic Munc13-1 levels, a similar reduction of the number of docked synaptic vesicles, and a more pronounced decrease of total synaptic vesicle number. At the physiological level, these RIM-ELKS-Munc13 hextuple KO cultures display a further decrease in the pool of release-ready synaptic vesicles with largely unchanged release probability compared with RIM-ELKS quadruple KO cultures.

    The data presented in the study are of high quality, and the generation of RIM-ELKS-Munc13 hextuple KO mouse cultures further demonstrates the feasibility of complex KO mouse models. A major question that remains to be addressed is if the release that remains in the absence of RIM and ELKS indeed mostly depends on Munc13.

  9. Version published to 10.7554/elife.79077 on eLife
  10. Version published to 10.1101/2022.04.01.486686v1 on bioRxiv