Kerstin Reim

β Phorbol Ester- and Diacylglycerol-Induced Augmentation of Transmitter Release Is Mediated by Munc13s and Not by PKCs

Munc13-1 is a presynaptic protein with an essential role in synaptic vesicle priming. It contains a diacylglycerol (DAG)/beta phorbol ester binding C(1) domain and is a potential target of the DAG second messenger pathway that may act in parallel with PKCs. Using genetically modified mice that express a DAG/beta phorbol ester binding-deficient Munc13-1(H567K) variant instead of the wild-type protein, we determined the relative contribution of PKCs and Munc13-1 to DAG/beta phorbol ester-dependent regulation of neurotransmitter release. We show that Munc13s are the main presynaptic DAG/beta phorbol ester receptors in hippocampal neurons. Modulation of Munc13-1 activity by second messengers via the DAG/beta phorbol ester binding C(1) domain is essential for use-dependent alterations of synaptic efficacy and survival.

Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming

Synaptic vesicles must be primed to fusion competence before they can fuse with the plasma membrane in response to increased intracellular Ca2+ levels. The presynaptic active zone protein Munc13-1 is essential for priming of glutamatergic synaptic vesicles in hippocampal neurons. However, a small subpopulation of synapses in any given glutamatergic nerve cell as well as all γ-aminobutyratergic (GABAergic) synapses are largely independent of Munc13-1. We show here that Munc13-2, the only Munc13 isoform coexpressed with Munc13-1 in hippocampus, is responsible for vesicle priming in Munc13-1 independent hippocampal synapses. Neurons lacking both Munc13-1 and Munc13-2 show neither evoked nor spontaneous release events, yet form normal numbers of synapses with typical ultrastructural features. Thus, the two Munc13 isoforms are completely redundant in GABAergic cells whereas glutamatergic neurons form two types of synapses, one of which is solely Munc13-1 dependent and lacks Munc13-2 whereas the other type employs Munc13-2 as priming factor. We conclude that Munc13-mediated vesicle priming is not a transmitter specific phenomenon but rather a general and essential feature of multiple fast neurotransmitter systems, and that synaptogenesis during development is not dependent on synaptic secretory activity.

Complexins Regulate a Late Step in Ca2+-Dependent Neurotransmitter Release

Synaptic vesicle fusion at synapses is triggered by increases in cytosolic Ca2+ levels. However, the identity of the Ca2+ sensor and the transduction mechanism of the Ca2+ trigger are unknown. We show that Complexins, stoichiometric components of the exocytotic core complex, are important regulators of transmitter release at a step immediately preceding vesicle fusion. Neurons lacking Complexins show a dramatically reduced transmitter release efficiency due to decreased Ca2+ sensitivity of the synaptic secretion process. Analyses of mutant neurons demonstrate that Complexins are acting at or following the Ca2+-triggering step of fast synchronous transmitter release by regulating the exocytotic Ca2+ sensor, its interaction with the core complex fusion machinery, or the efficiency of the fusion apparatus itself.

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.

Distinct domains of complexin I differentially regulate neurotransmitter release.

Complexins constitute a family of four synaptic high-affinity SNARE complex–binding proteins. They positively regulate a late, post-priming step in Ca2+-triggered synchronous neurotransmitter release, but the underlying molecular mechanisms are unclear. We show here that SNARE complex binding of complexin I (CplxI) via its central α-helix is necessary but, unexpectedly, not sufficient for its key function in promoting neurotransmitter release. An accessory α-helix on the N-terminal side of the SNARE complex–binding region has an inhibitory effect on fast synaptic exocytosis, whereas sequences N-terminally adjacent to this helix facilitate Ca2+-triggered release even in the absence of the Ca2+ sensor synaptotagmin-1. Our results indicate that distinct functional domains of CplxI differentially regulate synaptic exocytosis and that, through the interplay between these domains, CplxI carries out a crucial role in fine-tuning Ca2+-triggered fast neurotransmitter release.

Structurally and functionally unique complexins at retinal ribbon synapses

Ribbon synapses in retinal sensory neurons maintain large pools of readily releasable synaptic vesicles. This allows them to release several hundreds of vesicles per second at every presynaptic release site. The molecular components that cause this high transmitter release efficiency of ribbon synapses are unknown. In the present study, we identified and characterized two novel vertebrate complexins (CPXs), CPXs III and IV, that are the only CPX isoforms present in retinal ribbon synapses. CPXs III and IV are COOH-terminally farnesylated, and, like CPXs I and II, bind to SNAP receptor complexes. CPXs III and IV can functionally replace CPXs I and II, and their COOH-terminal farnesylation regulates their synaptic targeting and modulatory function in transmitter release. The novel CPXs III and IV may contribute to the unique release efficacy of retinal sensory neurons.

Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system

Complexins (Cplxs) are key regulators of synaptic exocytosis, but whether they act as facilitators or inhibitors is currently being disputed controversially. We show that genetic deletion of all Cplxs expressed in the mouse brain causes a reduction in Ca2+-triggered and spontaneous neurotransmitter release at both excitatory and inhibitory synapses. Our results demonstrate that at mammalian central nervous system synapses, Cplxs facilitate neurotransmitter release and do not simply act as inhibitory clamps of the synaptic vesicle fusion machinery.

CAPS1 Regulates Catecholamine Loading of Large Dense-Core Vesicles

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.

Novel Markers Reveal Subpopulations of Subplate Neurons in the Murine Cerebral Cortex

The subplate lays the foundation of the developing cerebral cortex, and abnormalities have been suggested to contribute to various brain developmental disorders. The causal relationship between cellular pathologies and cognitive disorders remains unclear, and therefore, a better understanding of the role of subplate cells in cortical development is essential. Only by determining the molecular taxonomy of this diverse class of neurons can we identify the subpopulations that may contribute differentially to cortical development. We identified novel markers for murine subplate cells by comparing gene expression of subplate and layer 6 of primary visual and somatosensory cortical areas of postnatal day (P)8 old mice using a microarray-based approach. We examined the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) where the subplate is displaced in relation to the cortical plate. In situ hybridization or immunohistochemistry confirmed subplate-selective expression of complexin 3, connective tissue growth factor, nuclear receptor-related 1/Nr4a2, and monooxygenase Dbh-like 1 while transmembrane protein 163 also had additional expression in layer 5, and DOPA decarboxylase was also present in the white matter. Localization of marker-positive cells in the reeler and p35-KO cortices suggests different subpopulations of subplate cells. These new markers open up possibilities for further identification of subplate subpopulations in research and in neuropathological diagnosis.