Roger Janz

Rab3A is essential for mossy fibre long-term potentiation in the hippocampus

Repetitive activation of excitatory synapses in the central nervous system results in a long-lasting increase in synaptic transmission called long-term potentiation (LTP). It is generally believed that this synaptic plasticity may underlie certain forms of learning and memory. LTP at most synapses involves the activation of the NMDA (N-methyl-d-aspartate) subtype of glutamate receptor, but LTP at hippocampal mossy fibre synapses is independent of NMDA receptors and has a component that is induced and expressed presynaptically. It appears to be triggered by a rise in presynaptic Ca2+(refs 2, 3), and requires the activation of protein kinase A, which leads to an increased release of glutamate,. Agreat deal is known about the biochemical steps involved in the vesicular release of transmitter, but none of these steps has been directly implicated in long-term synaptic plasticity. Here we show that, although a variety of short-term plasticities are normal, LTP at mossy fibre synapses is abolished in mice lacking the synaptic vesicle protein Rab3A.

SV2A and SV2B Function as Redundant Ca2+ Regulators in Neurotransmitter Release

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor isoform (SV2C) that resemble transporter proteins. We now show that SV2B knockout mice are phenotypically normal while SV2A- and SV2A/SV2B double knockout mice exhibit severe seizures and die postnatally. In electrophysiological recordings from cultured hippocampal neurons, SV2A- or SV2B-deficient cells exhibited no detectable abnormalities. Neurons lacking both SV2 isoforms, however, experienced sustained increases in Ca2+-dependent synaptic transmission when two or more action potentials were triggered in succession. These increases could be reversed by EGTA-AM. Our data suggest that without SV2 proteins, presynaptic Ca2+ accumulation during consecutive action potentials causes abnormal increases in neurotransmitter release that destabilize synaptic circuits and induce epilepsy.

Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I

We have generated mice lacking synaptogyrin I and synaptophysin I to explore the functions of these abundant tyrosine-phosphorylated proteins of synaptic vesicles. Single and double knockout mice were alive and fertile without significant morphological or biochemical changes. Electrophysiological recordings in the hippocampal CA1 region revealed that short-term and long-term synaptic plasticity were severely reduced in the synaptophysin/synaptogyrin double knockout mice. LTP was decreased independent of the induction protocol, suggesting that the defect in LTP was not caused by insufficient induction. Our data show that synaptogyrin I and synaptophysin I perform redundant and essential functions in synaptic plasticity without being required for neurotransmitter release itself.

Glycosylated SV2A and SV2B Mediate the Entry of Botulinum Neurotoxin E into Neurons

Botulinum neurotoxin E (BoNT/E) can cause paralysis in humans and animals by blocking neurotransmitter release from presynaptic nerve terminals. How this toxin targets and enters neurons is not known. Here we identified two isoforms of the synaptic vesicle protein SV2, SV2A and SV2B, as the protein receptors for BoNT/E. BoNT/E failed to enter neurons cultured from SV2A/B knockout mice; entry was restored by expressing SV2A or SV2B, but not SV2C. Mice lacking SV2B displayed reduced sensitivity to BoNT/E. The fourth luminal domain of SV2A or SV2B alone, expressed in chimeric receptors by replacing the extracellular domain of the low-density lipoprotein receptor, can restore the binding and entry of BoNT/E into neurons lacking SV2A/B. Furthermore, we found disruption of a N-glycosylation site (N573Q) within the fourth luminal domain of SV2A rendered the mutant unable to mediate the entry of BoNT/E and also reduced the entry of BoNT/A. Finally, we demonstrate that BoNT/E failed to bind and enter ganglioside-deficient neurons; entry was rescued by loading exogenous gangliosides into neuronal membranes. Together, the data reported here demonstrate that glycosylated SV2A and SV2B act in conjunction with gangliosides to mediate the entry of BoNT/E into neurons.

Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics

Silencing neurons using optogenetics Rhodopsin light-sensitive ion channels from green algae provide a powerful tool to control neuronal circuits. Rhodopsin cation channels effectively depolarize neurons and cause the firing of short-lived electrical membrane potentials. Govorunova et al. describe algal channels that do the opposite; that is, they hyperpolarize or silence particular neurons (see the Perspective by Berndt and Deisseroth). These cation channels provide greater light sensitivity than that of existing hyperpolarizing light-activated channels, operate rapidly, and selectively conduct only anions. This approach is an ideal complement to the widely used technique of creating light-sensitive neurons through the expression of rhodopsin cation channels. Science, this issue p. 647; see also p. 590 An anion channel from algae allows optogenetic silencing of neurons. [Also see Perspective by Berndt and Deisseroth] Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

A Role for cAMP in Long-Term Depression at Hippocampal Mossy Fiber Synapses

Mossy fiber synapses on hippocampal CA3 pyramidal cells, in addition to expressing an NMDA receptor-independent form of long-term potentiation (LTP), have recently been shown to express a novel presynaptic form of long-term depression (LTD). We have studied the mechanisms underlying mossy fiber LTD and present evidence that it is triggered, at least in part, by a metabotropic glutamate receptor-mediated decrease in adenylyl cyclase activity, which leads to a decrease in the activity of the cAMP-dependent protein kinase (PKA) and a reversal of the presynaptic processes responsible for mossy fiber LTP. The bidirectional control of synaptic strength at mossy fiber synapses by activity therefore appears to be due to modulation of the cAMP-PKA signaling pathway in mossy fiber boutons.

SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family

We describe here the identification and molecular characterization of a new brain protein that we named SV2C because it is homologous to the synaptic vesicle proteins SV2A and SV2B, and because it is also recognized by the monoclonal SV2 antibody that led to the initial discovery of SV2A and SV2B. SV2C is more closely related to SV2A (62% identity) than to SV2B (57% identity), and contains 12 transmembrane regions similar to these proteins. To characterize SV2C and compare its properties and localization with those of SV2A and SV2B, we raised an SV2C-specific antibody. Using this antibody, we show that SV2C is an N-glycosylated protein that is concentrated on small synaptic vesicles; in addition, it is found on microvesicles in adrenal chromaffin cells. We evaluated the relative localization of the three SV2 isoforms by staining rat brain sections with antibodies specific for SV2A, SV2B and SV2C. Analysis of the resulting staining patterns confirmed previous conclusions that SV2A is ubiquitously expressed in virtually all synapses. SV2B, although more restricted in distribution, was also found in a wide variety of synapses throughout the brain. In striking contrast to this general localization and to similarly wide distributions of other synaptic vesicle proteins, SV2C was observed only in few brain areas. High levels of SV2C were found primarily in phylogenetically old brain regions such as the pallidum, the substantia nigra, the midbrain, the brainstem and the olfactory bulb. SV2C was undetectable in the cerebral cortex and the hippocampus, and found at low levels in the cerebellar cortex. Our data suggest that closely related members of a synaptic vesicle protein family can either have very general (SV2A) or restricted distributions (SV2C), possibly in order to allow specialization in the regulation of the expression or of the function of these abundant synaptic vesicle proteins.

Mechanism of Action of rab3A in Mossy Fiber LTP

In mossy fiber synapses of the hippocampal CA3 region, LTP is induced by cAMP and requires the synaptic vesicle protein rab3A. In contrast, CA1-region synapses do not exhibit this type of LTP. We now show that cAMP enhances glutamate release from CA3 but not CA1 synaptosomes by (1) increasing the readily releasable pool as tested by hypertonic sucrose; (2) potentiating release evoked by KCl depolarization, which opens voltage-gated Ca2+ channels; and (3) by enhancing Ca2+ action on the secretory apparatus as monitored by the Ca2+-ionophore ionomycin. In rab3A-deficient synaptosomes, forskolin still enhances KCl- and sucrose-induced glutamate release but not ionomycin-induced release. Our results show that cAMP has multiple actions in mossy fiber synapses, of which only the direct activation of the secretory apparatus requires rab3A and functions in mfLTP.

Physical and Functional Interaction between the Dopamine Transporter and the Synaptic Vesicle Protein Synaptogyrin-3

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein–protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Synaptogyrins Regulate Ca2+-dependent Exocytosis in PC12 Cells

Synaptogyrins constitute a family of synaptic vesicle proteins of unknown function. With the full-length structure of a new brain synaptogyrin isoform, we now show that the synaptogyrin family in vertebrates includes two neuronal and one ubiquitous isoform. All of these synaptogyrins are composed of a short conserved N-terminal cytoplasmic sequence, four homologous transmembrane regions, and a variable cytoplasmic C-terminal tail that is tyrosine-phosphorylated. The localization, abundance, and conservation of synaptogyrins suggest a function in exocytosis. To test this, we employed a secretion assay in PC12 cells expressing transfected human growth hormone (hGH) as a reporter protein. When Ca2+-dependent hGH secretion from PC12 cells was triggered by high K+ or α-latrotoxin, co-transfection of all synaptogyrins with hGH inhibited hGH exocytosis as strongly as co-transfection of tetanus toxin light chain. Synaptophysin I, which is distantly related to synaptogyrins, was also inhibitory but less active. Inhibition was independent of the amount of hGH expressed but correlated with the amount of synaptogyrin transfected. Inhibition of exocytosis was not observed with several other synaptic proteins, suggesting specificity. Analysis of the regions of synaptogyrin required for inhibition revealed that the conserved N-terminal domain of synaptogyrin is essential for inhibition, whereas the long C-terminal cytoplasmic tail is largely dispensable. Our results suggest that synaptogyrins are conserved components of the exocytotic apparatus, which function as regulators of Ca2+-dependent exocytosis.