Arthur P.H. de Jong

Doc2b is a High Affinity Ca2+ Sensor for Spontaneous Neurotransmitter Release

“Spontaneous” Release Trigger Synaptic vesicle release occurs in different phases that can be tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events not triggered by action potentials (spontaneous). The vesicle protein synaptotagmin is thought to act as the Ca2+ sensor in the synchronous phase, but for the other two phases, Ca2+ sensors have not been identified. Groffen et al. (p. 1614, published online 11 February) now show that cytoplasmic proteins known as Doc2 (double C2 domain) proteins are required for spontaneous release. Doc2 proteins promote membrane fusion in response to exceptionally low increases in Ca2+, and are several orders of magnitude more sensitive to Ca2+ than synaptotagmin. Doc2 and synaptotagmin compete for SNARE-complex binding during membrane fusion. A mutation that abolishes the Ca2+ dependence of Doc2b also abolishes the Ca2+ dependence of spontaneous release. Thus, Doc2 is a high-affinity Ca2+ sensor for spontaneous release that competes with synaptotagmin for SNARE complex binding. Spontaneous synaptic vesicle fusion is triggered by soluble proteins that compete with synaptotagmins to induce membrane curvature. Synaptic vesicle fusion in brain synapses occurs in phases that are either tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events in the absence of action potentials (spontaneous). Synaptotagmin-1, -2, and -9 are vesicle-associated Ca2+ sensors for synchronous release. Here we found that double C2 domain (Doc2) proteins act as Ca2+ sensors to trigger spontaneous release. Although Doc2 proteins are cytosolic, they function analogously to synaptotagmin-1 but with a higher Ca2+ sensitivity. Doc2 proteins bound to N-ethylmaleimide–sensitive factor attachment receptor (SNARE) complexes in competition with synaptotagmin-1. Thus, different classes of multiple C2 domain–containing molecules trigger synchronous versus spontaneous fusion, which suggests a general mechanism for synaptic vesicle fusion triggered by the combined actions of SNAREs and multiple C2 domain–containing proteins.

Functional Gene Group Analysis Reveals a Role of Synaptic Heterotrimeric G Proteins in Cognitive Ability

Although cognitive ability is a highly heritable complex trait, only a few genes have been identified, explaining relatively low proportions of the observed trait variation. This implies that hundreds of genes of small effect may be of importance for cognitive ability. We applied an innovative method in which we tested for the effect of groups of genes defined according to cellular function (functional gene group analysis). Using an initial sample of 627 subjects, this functional gene group analysis detected that synaptic heterotrimeric guanine nucleotide binding proteins (G proteins) play an important role in cognitive ability (PEMP = 1.9 × 10−4). The association with heterotrimeric G proteins was validated in an independent population sample of 1507 subjects. Heterotrimeric G proteins are central relay factors between the activation of plasma membrane receptors by extracellular ligands and the cellular responses that these induce, and they can be considered a point of convergence, or a “signaling bottleneck.” Although alterations in synaptic signaling processes may not be the exclusive explanation for the association of heterotrimeric G proteins with cognitive ability, such alterations may prominently affect the properties of neuronal networks in the brain in such a manner that impaired cognitive ability and lower intelligence are observed. The reported association of synaptic heterotrimeric G proteins with cognitive ability clearly points to a new direction in the study of the genetic basis of cognitive ability.

Automated analysis of neuronal morphology, synapse number and synaptic recruitment

The shape, structure and connectivity of nerve cells are important aspects of neuronal function. Genetic and epigenetic factors that alter neuronal morphology or synaptic localization of pre- and post-synaptic proteins contribute significantly to neuronal output and may underlie clinical states. To assess the impact of individual genes and disease-causing mutations on neuronal morphology, reliable methods are needed. Unfortunately, manual analysis of immuno-fluorescence images of neurons to quantify neuronal shape and synapse number, size and distribution is labor-intensive, time-consuming and subject to human bias and error. We have developed an automated image analysis routine using steerable filters and deconvolutions to automatically analyze dendrite and synapse characteristics in immuno-fluorescence images. Our approach reports dendrite morphology, synapse size and number but also synaptic vesicle density and synaptic accumulation of proteins as a function of distance from the soma as consistent as expert observers while reducing analysis time considerably. In addition, the routine can be used to detect and quantify a wide range of neuronal organelles and is capable of batch analysis of a large number of images enabling high-throughput analysis.

Presynaptic signal transduction pathways that modulate synaptic transmission.

Presynaptic modulation is a crucial factor in the adaptive capacity of the nervous system. The coupling between incoming action potentials and neurotransmitter secretion is modulated by firstly, recent activity of the presynaptic axon that leads to the accumulation of residual calcium in the terminal and secondly, activation of presynaptic receptors by external signals. Despite the detailed description of these phenomena, the underlying mechanisms are still poorly understood. The nerve terminal contains many Ca(2+)-binding proteins that may contribute to the translation of residual Ca(2+)-increases to secretion modulation. We also found that >100 presynaptic proteins are phosphorylated and may contribute to the translation of presynaptic receptor activation to secretion modulation. However, which of these many candidates are the dominant regulators and how their activities integrate is largely unknown. Here, we review some of the recent insights into the complex interplay between presynaptic signal transduction components and propose blueprints of the major pathways.

Munc13 controls the location and efficiency of dense-core vesicle release in neurons

Although Munc13-1 and Munc13-2 facilitate dense-core vesicle fusion, they are not required for DCV release, in contrast to their essential role in synaptic vesicle exocytosis.

CCL21-induced calcium transients and proliferation in primary mouse astrocytes: CXCR3-dependent and independent responses

CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in damaged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10(-9) M and 10(-8) M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high concentrations of CCL21 (10(-7) M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 promoted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (chemokine-) receptor, which is activated at high CCL21 concentrations.

Translating neuronal activity at the synapse: presynaptic calcium sensors in short-term plasticity

The complex manner in which patterns of presynaptic neural activity are translated into short-term plasticity (STP) suggests the existence of multiple presynaptic calcium (Ca2+) sensors, which regulate the amplitude and time-course of STP and are the focus of this review. We describe two canonical Ca2+-binding protein domains (C2 domains and EF-hands) and define criteria that need to be met for a protein to qualify as a Ca2+ sensor mediating STP. With these criteria in mind, we discuss various forms of STP and identify established and putative Ca2+ sensors. We find that despite the multitude of proposed sensors, only three are well established in STP: Munc13, protein kinase C (PKC) and synaptotagmin-7. For putative sensors, we pinpoint open questions and potential pitfalls. Finally, we discuss how the molecular properties and modes of action of Ca2+ sensors can explain their differential involvement in STP and shape net synaptic output.

Dendritic position is a major determinant of presynaptic strength

Positional information, independent of neuronal activity, regulates presynaptic strength, with the strongest presynaptic terminals closest to the soma.

Protein kinase C is a calcium sensor for presynaptic short-term plasticity

In presynaptic boutons, calcium (Ca2+) triggers both neurotransmitter release and short-term synaptic plasticity. Whereas synaptotagmins are known to mediate vesicle fusion through binding of high local Ca2+ to their C2 domains, the proteins that sense smaller global Ca2+ increases to produce short-term plasticity have remained elusive. Here, we identify a Ca2+ sensor for post-tetanic potentiation (PTP), a form of plasticity thought to underlie short-term memory. We find that at the functionally mature calyx of Held synapse the Ca2+-dependent protein kinase C isoforms α and β are necessary for PTP, and the expression of PKCβ in PKCαβ double knockout mice rescues PTP. Disruption of Ca2+ binding to the PKCβ C2 domain specifically prevents PTP without impairing other PKCβ-dependent forms of synaptic enhancement. We conclude that different C2-domain-containing presynaptic proteins are engaged by different Ca2+ signals, and that Ca2+ increases evoked by tetanic stimulation are sensed by PKCβ to produce PTP. DOI: http://dx.doi.org/10.7554/eLife.03011.001

RIM C2B Domains Target Presynaptic Active Zone Functions to PIP2-Containing Membranes

Rapid and efficient synaptic vesicle fusion requires a pool of primed vesicles, the nearby tethering of Ca2+ channels, and the presence of the phospholipid PIP2 in the target membrane. Although the presynaptic active zone mediates the first two requirements, it is unclear how fusion is targeted to membranes with high PIP2 content. Here we find that the C2B domain of the active zone scaffold RIM is critical for action potential-triggered fusion. Remarkably, the known RIM functions in vesicle priming and Ca2+ influx do not require RIM C2B domains. Instead, biophysical experiments reveal that RIM C2 domains, which lack Ca2+ binding, specifically bind to PIP2. Mutational analyses establish that PIP2 binding to RIM C2B and its tethering to the other RIM domains are crucial for efficient exocytosis. We propose that RIM C2B domains are constitutive PIP2-binding modules that couple mechanisms for vesicle priming and Ca2+ channel tethering to PIP2-containing target membranes.