Frédéric Doussau

How botulinum and tetanus neurotoxins block neurotransmitter release

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.

Structural Domains Involved in the Regulation of Transmitter Release by Synapsins

Synapsins are a family of neuron-specific phosphoproteins that regulate neurotransmitter release by associating with synaptic vesicles. Synapsins consist of a series of conserved and variable structural domains of unknown function. We performed a systematic structure-function analysis of the various domains of synapsin by assessing the actions of synapsin fragments on neurotransmitter release, presynaptic ultrastructure, and the biochemical interactions of synapsin. Injecting a peptide derived from domain A into the squid giant presynaptic terminal inhibited neurotransmitter release in a phosphorylation-dependent manner. This peptide had no effect on vesicle pool size, synaptic depression, or transmitter release kinetics. In contrast, a peptide fragment from domain C reduced the number of synaptic vesicles in the periphery of the active zone and increased the rate and extent of synaptic depression. This peptide also slowed the kinetics of neurotransmitter release without affecting the number of docked vesicles. The domain C peptide, as well as another peptide from domain E that is known to have identical effects on vesicle pool size and release kinetics, both specifically interfered with the binding of synapsins to actin but not with the binding of synapsins to synaptic vesicles. This suggests that both peptides interfere with release by preventing interactions of synapsins with actin. Thus, interactions of domains C and E with the actin cytoskeleton may allow synapsins to perform two roles in regulating release, whereas domain A has an actin-independent function that regulates transmitter release in a phosphorylation-sensitive manner.

Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge

Significance The inferior olive, one of the major source of inputs to the cerebellum, sends climbing fibers to Purkinje cells, the key processing units of cerebellar-dependent motor control. Using an optogenetic strategy, we demonstrate that Purkinje cells disinhibit their climbing-fiber afferents via a poly-synaptic circuit. These findings identify a functional closed-loop organization in the olivo-cerebellar circuits that is potentially important for cerebellar motor learning. Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning.

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.

Adaptation of granule cell to Purkinje cell synapses to high-frequency transmission.

The mossy fiber (MF)–granule cell (GC) pathway conveys multiple modalities of information to the cerebellar cortex, converging on Purkinje cells (PC), the sole output of the cerebellar cortex. Recent in vivo experiments have shown that activity in GCs varies from tonic firing at a few hertz to phasic bursts >500 Hz. However, the responses of parallel fiber (PF)–PC synapses to this wide range of input frequencies are unknown, and there is controversy regarding several frequency-related parameters of transmission at this synapse. We performed recordings of unitary synapses and combined variance–mean analysis with a carefully adapted extracellular stimulation method in young and adult rats. We show that, although the probability of release at individual sites is low at physiological calcium concentration, PF–PC synapses release one or more vesicles with a probability of 0.44 at 1.5 mm [Ca2+]e. Paired-pulse facilitation was observed over a wide range of frequencies; it renders burst inputs particularly effective and reproducible. These properties are primarily independent of synaptic weight and age. Furthermore, we show that the PF–PC synapse is able to sustain transmission at very high frequencies for tens of stimuli, as a result of accelerated vesicle replenishment and an apparent recruitment of release site vesicles, which appears to be a central mechanism of paired-pulse facilitation at this synapse. These properties ensure that PF–PC synapses possess a dynamic range enabling the temporal code of MF inputs to be transmitted reliably to the PC.

Late-Life Environmental Enrichment Induces Acetylation Events and Nuclear Factor κB-Dependent Regulations in the Hippocampus of Aged Rats Showing Improved Plasticity and Learning.

Aging weakens memory functions. Exposing healthy rodents or pathological rodent models to environmental enrichment (EE) housing improves their cognitive functions by changing neuronal levels of excitation, cellular signaling, and plasticity, notably in the hippocampus. At the molecular level, brain derived-neurotrophic factor (BDNF) represents an important player that supports EE-associated changes. EE facilitation of learning was also shown to correlate with chromatin acetylation in the hippocampus. It is not known, however, whether such mechanisms are still into play during aging. In this study, we exposed a cohort of aged rats (18-month-old) to either a 6 month period of EE or standard housing conditions and investigated chromatin acetylation-associated events [histone acetyltranferase activity, gene expression, and histone 3 (H3) acetylation] and epigenetic modulation of the Bdnf gene under rest conditions and during learning. We show that EE leads to upregulation of acetylation-dependent mechanisms in aged rats, whether at rest or following a learning challenge. We found an increased expression of Bdnf through Exon-I-dependent transcription, associated with an enrichment of acetylated H3 at several sites of Bdnf promoter I, more particularly on a proximal nuclear factor κB (NF-κB) site under learning conditions. We further evidenced p65/NF-κB binding to chromatin at promoters of genes important for plasticity and hippocampus-dependent learning (e.g., Bdnf, CamK2D). Altogether, our findings demonstrate that aged rats respond to a belated period of EE by increasing hippocampal plasticity, together with activating sustained acetylation-associated mechanisms recruiting NF-κB and promoting related gene transcription. These responses are likely to trigger beneficial effects associated with EE during aging. SIGNIFICANCE STATEMENT Aging weakens memory functions. Optimizing the neuronal circuitry required for normal brain function can be achieved by increasing sensory, motor, and cognitive stimuli resulting from interactions with the environment (behavioral therapy). This can be experimentally modeled by exposing rodents to environmental enrichment (EE), as with large cages, numerous and varied toys, and interaction with other rodents. However, EE effects in aged rodents has been poorly studied, and it is not known whether beneficial mechanisms evidenced in the young adults can still be recruited during aging. Our study shows that aged rats respond to a belated period of EE by activating specific epigenetic and transcriptional signaling that promotes gene expression likely to facilitate plasticity and learning behaviors.

Fast changes in the functional status of release sites during short‐term plasticity: involvement of a frequency‐dependent bypass of Rac at Aplysia synapses

Synaptic transmission can be described as a stochastic quantal process defined by three main parameters: N, the number of functional release sites; P, the release probability; and Q, the quantum of response. Many changes in synaptic strength that are observed during expression of short term plasticity rely on modifications in P. Regulation of N has been also suggested. We have investigated at identified cholinergic inhibitory Aplysia synapses the cellular mechanism of post‐tetanic potentiation (PTP) expressed under control conditions or after N has been depressed by applying lethal toxin (LT) from Clostridium sordellii or tetanus toxin (TeNT). The analysis of the Ca2+ dependency, paired‐pulse ratio and variance to mean amplitude relationship of the postsynaptic responses elicited at distinct extracellular [Ca2+]/[Mg2+] elicited during control post‐tetanic potentiation (PTPcont) indicated that PTPcont is mainly driven by an increase in release probability, P. The PTP expressed at TeNT‐treated synapses (PTPTeNT) was found to be similar to PTPcont, but scaled to the extent of reduction in N produced by TeNT. Despite LT inducing a decrease in N as TeNT does, the PTP expressed at LT‐treated synapses (PTPLT) was characterized by exceptionally large amplitude and bi‐exponential time course, as compared to PTPcont or the PTPTeNT. Analysis of the Ca2+ dependency of PTPLT, paired‐pulse ratio and fluctuations in amplitude of the postsynaptic responses elicited during PTPLT or the variance to mean amplitude relationship of time‐locked postsynaptic responses in a series of subsequent PTPLT indicated that an N‐driven change is involved in the early phase (1 s time scale) of PTPLT, while at a later stage PTPLT is composed of both N and P increases. Our results suggest that fast switching on of the functional status of the release sites occurs also during the early events of PTPcont. The early N‐driven phase of PTPLT is likely to be a functional recovery of the release sites silenced by Rac inactivation. This effect did not appear to result from reversion of LT inhibitory action but from bypassing the step regulated by Rac. Altogether the data suggest that Rac and the intracellular pathway which allows the bypassing of Rac are key players in new forms of short‐term plasticity that rely on fast, activity‐dependent changes in the functional status of the release sites.

Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination

Epsilon toxin (ET) is produced by Clostridium perfringens types B and D and causes severe neurological disorders in animals. ET has been observed binding to white matter, suggesting that it may target oligodendrocytes. In primary cultures containing oligodendrocytes and astrocytes, we found that ET (10−9u2009M and 10−7u2009M) binds to oligodendrocytes, but not to astrocytes. ET induces an increase in extracellular glutamate, and produces oscillations of intracellular Ca2+ concentration in oligodendrocytes. These effects occurred without any change in the transmembrane resistance of oligodendrocytes, underlining that ET acts through a pore‐independent mechanism. Pharmacological investigations revealed that the Ca2+ oscillations are caused by the ET‐induced rise in extracellular glutamate concentration. Indeed, the blockade of metabotropic glutamate receptors type 1 (mGluR1) prevented ET‐induced Ca2+ signals. Activation of the N‐methyl‐D‐aspartate receptor (NMDA‐R) is also involved, but to a lesser extent. Oligodendrocytes are responsible for myelinating neuronal axons. Using organotypic cultures of cerebellar slices, we found that ET induced the demyelination of Purkinje cell axons within 24u2009h. As this effect was suppressed by antagonizing mGluR1 and NMDA‐R, demyelination is therefore caused by the initial ET‐induced rise in extracellular glutamate concentration. This study reveals the novel possibility that ET can act on oligodendrocytes, thereby causing demyelination. Moreover, it suggests that for certain cell types such as oligodendrocytes, ET can act without forming pores, namely through the activation of an undefined receptor‐mediated pathway.

A Novel Form of Presynaptic Plasticity Based on the Fast Reactivation of Release Sites Switched Off during Low-Frequency Depression

Repetitive firing of neurons at a low frequency often leads to a decrease in synaptic strength. The mechanism of this low-frequency depression (LFD) is poorly understood. Here, LFD was studied at Aplysia cholinergic synapses. The absence of a significant change in the paired-pulse ratio during LFD, together with the facts that neither the time course nor the extent of LFD were affected by the initial release probability, suggests that LFD is not related to a depletion of the ready-to-fuse synaptic vesicles (SVs) or to a decrease in the release probability, but results from the silencing of a subpopulation of release sites. A subset of SVs or release sites, which acquired a high release probability status during LFD, permits synapses to rapidly and temporarily recover the initial synaptic strength when the stimulation is stopped. However, the recovery of the full capacity of the synapse to sustain repetitive stimulations is slow and involves spontaneous reactivation of the silent release sites. Application of tetanic stimulations accelerates this recovery by immediately switching on the silent sites. This high-frequency-dependent phenomenon underlies a new form of synaptic plasticity that allows resetting of presynaptic efficiency independently of the recent history of the synapse. Microinjection of a mutated Aplysia synapsin that cannot be phosphorylated by cAMP-dependent protein kinase (PKA), or a PKA inhibitor both prevented high-frequency-dependent awakening of release sites. Changes in the firing pattern of neurons appear to be able to regulate the on–off status of release sites via a molecular cascade involving PKA-dependent phosphorylation of synapsin.

Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function.

Inhibition of neuroexocytosis by tetanus neurotoxin (TeNT) involves VAMP-2/synaptobrevin-2 cleavage. However, deletion of the TeNT activity does not completely abolish its inhibitory action. TeNT is a potent activator of the cross-linking enzyme transglutaminase 2 (TGase 2) in vitro. The role of the latter mechanism in TeNT poisoning was investigated in isolated nerve terminals and intact neurons. TeNT-induced inhibition of glutamate release from rat cortical synaptosomes was associated with a simultaneous activation of neuronal transglutaminase (TGase) activity. The TeNT-induced blockade of neuroexocytosis was strongly attenuated by pretreatment of either live Aplysia neurons or isolated nerve terminals with specific TGase inhibitors or neutralizing antibodies. The same treatments completely abolished the residual blockade of neuroexocytosis of a non-proteolytic mutant of TeNT light chain. Electrophysiological studies indicated that TGase activation occurs at an early step of TeNT poisoning and contributes to the inhibition of transmitter release. Bioinformatics and biochemical analyses identified synapsin I and SNAP-25 as potential presynaptic TGase substrates in isolated nerve terminals, which are potentially involved in the inhibitory action of TeNT. The results suggest that neuronal TGase activity plays an important role in the regulation of neuroexocytosis and is one of the intracellular targets of TeNT in neurons.