Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons

Abstract

Tetanus neurotoxin causes the spastic paralysis of tetanus by blocking neurotransmitter release at inhibitory synapses of the spinal cord. This is due to the penetration of the toxin inside the neuronal cytosol where it cleaves specifically VAMP͞synaptobrevin, an essential component of the neuroexocytosis apparatus. Here we show that tetanus neurotoxin is internalized inside the lumen of small synaptic vesicles following the process of vesicle reuptake. Vesicle acidification is essential for the toxin translocation in the cytosol, which results in the proteolytic cleavage of VAMP͞ synaptobrevin and block of exocytosis.Upon membrane depolarization and calcium entry into nerve terminals, synaptic vesicles (SVs) docked at active zones release their neurotransmitter content into the synaptic cleft (1-4). SVs are then rapidly recycled in an endocytic process that requires dynamin and clathrin (5, 6). The biochemical steps involved in vesicle docking, fusion, and reuptake are actively investigated. Recently, several protein components of the neuroexocytosis apparatus have been identified (4, 7-9), and a model for docking and fusion of vesicles with target membranes has been proposed (10). One of these proteins, a SV membrane protein, VAMP͞ synaptobrevin, is the specific target of the metalloproteinase activity of tetanus neurotoxin (TeNT) (11,12). This neurotoxin consists of two disulfide-linked polypeptide chains: H (100 kDa) mediates the specific binding to the neuronal presynaptic membrane, while L (50 kDa) is responsible for the intracellular proteolytic activity. The spastic paralysis of tetanus is due to the specific intoxication of the spinal cord inhibitory interneurons by TeNT, which penetrates these cells and blocks the release of inhibitory neurotransmitters (13)(14)(15)(16). In vitro TeNT is active also on other central nervous system (CNS) neurons: it inhibits ␥-aminobutyrate (GABA)-mediated effects in rat hippocampal slices (17) and, when injected in rat ventral hippocampi, it causes an epileptiform syndrome (18,19).The steps involved in TeNT entry and trafficking inside peripheral and central neurons are not known. It is well documented that any form of nerve stimulation, and hence increase in synaptic activity, in the animal or in the phrenic nerve hemidiaphragm preparation, decreases the time of the onset of paralysis (20)(21)(22)(23)(24). Indirect evidence suggest that TeNT intoxication of spinal cord neurons involves TeNT transit through an intracellular acidic compartment (25,26). To investigate the route of entry of TeNT inside CNS neurons, we have used primary cultures of hippocampal neurons, which represent a wellcharacterized experimental model (27). These neurons develop in vitro through a stereotyped sequence of events and eventually they form in vitro functional synaptic contacts, characterized by an active SV recycling (28,29). SVs clustered at presynaptic sites of cultured hippocampal neurons internalize antibodies to the intravesicular domain of the SV protein synaptotagmin in a d...

Tetanus Toxin Is Transported in a Novel Neuronal Compartment Characterized by a Specialized pH Regulation

“…In fact, acidic pH triggers a conformational change of TeNT, which allows its membrane insertion and the translocation of the L chain into the cytosol, where it cleaves VAMP (2). This phenomenon occurs in hippocampal neurons in vitro (27) and in spinal cord inhibitory interneurons in vivo (1,28). In artificial liposomes, the pH initiating the membrane insertion of TeNT is ϳ5.0 (29), which is within the pH range of the endosomal pathway.…”

Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine

“…5). In neurons, the effects of TeNT depend on receptormediated endocytosis of the toxin and the subsequent release of the active subunit into the cytoplasm (37). Prolonged incubations and high toxin concentration are required to inhibit glutamate or ATP release from cultured astrocytes (19,21,38).…”

On the Role of Voltage-Dependent Calcium Channels in Calcium Signaling of AstrocytesIn Situ

“…Accordingly, the [Ca 2ϩ ] i decrease caused by depolarization likely depends on a decrease in the driving force for Ca 2ϩ influx. As to the second, TeNT is known to be highly neuron-specific and to exert its action on VAMP/synaptobrevin, a protein that plays a crucial role in the neuroexocytosis process (Schiavo et al, 1992;Matteoli et al, 1996). The inhibitory effect of the neurotoxin on neurotransmitter exocytosis was confirmed in each slice by the observations that (1) electrical stimulation of Schaffer collaterals failed to induce [Ca 2ϩ ] i increases in hippocampal neurons otherwise observed in TeNT-untreated slices; (2) pyramidal neurons still responded to K ϩ -induced depolarization with an early [Ca 2ϩ ] i increase, but the second [Ca 2ϩ ] i peak in their response was either greatly reduced or abolished; and (3) [Ca 2ϩ ] i basal levels were recovered much faster than in neurons from TeNT-untreated slices.…”