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Transient silencing of synaptic transmitter release from specific neuronal types by recombinant tetanus toxin light chain fused to antibody variable region.

ABSTRACT: We developed a novel strategy for conditional silencing of synaptic transmission in specific neuronal types in transgenic animals. We generated a recombinant protein termed immuno-tetanus toxin (ITet), which contains a monoclonal antibody variable region for human interleukin-2 receptor alpha-subunit (IL-2Ralpha) fused to tetanus toxin light chain. ITet was designed to transiently suppress transmitter release from target neurons genetically engineered to express human IL-2Ralpha via proteolytic cleavage of vesicle-associated membrane protein-2 (VAMP-2). The in vivo actions of ITet were investigated by using mutant mice that express IL-2Ralpha in striatal neurons under the control of the gene encoding dopamine D(2) receptor. Unilateral ITet injection into the striatum induced rotational behavior in the mutant mice and the rotations gradually reversed to the normal level. The behavioral alteration was accompanied by a transient decrease in the striatal VAMP-2 level and depolarization-evoked transmitter release in synaptic target region. However, ITet injection caused no structural change in striatal cells and nerve terminals in the mutants. These data indicate that ITet acts on striatal neurons bearing human IL-2Ralpha and temporally reduces their VAMP-2 content, thereby causing the blockade of transmitter release. Our ITet technology provides a useful approach for inducible and reversible control of synaptic transmission in specific neuronal types in the brain.

SUBMITTER: Kobayashi T

PROVIDER: S-EPMC2591090 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Transient silencing of synaptic transmitter release from specific neuronal types by recombinant tetanus toxin light chain fused to antibody variable region.

Kobayashi Tomoko T Kai Nobuyuki N Kobayashi Kenta K Fujiwara Tomonori T Akagawa Kimio K Onda Masanori M Kobayashi Kazuto K

Journal of neuroscience methods 20080819 1

We developed a novel strategy for conditional silencing of synaptic transmission in specific neuronal types in transgenic animals. We generated a recombinant protein termed immuno-tetanus toxin (ITet), which contains a monoclonal antibody variable region for human interleukin-2 receptor alpha-subunit (IL-2Ralpha) fused to tetanus toxin light chain. ITet was designed to transiently suppress transmitter release from target neurons genetically engineered to express human IL-2Ralpha via proteolytic ...[more]

PMID: 18775748

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Project description:The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, ?-lactamase (?lac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting ?15 and ?16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, ?-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (?lac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between ?15 and ?16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

Dataset Information

Transient silencing of synaptic transmitter release from specific neuronal types by recombinant tetanus toxin light chain fused to antibody variable region.

Publications

Transient silencing of synaptic transmitter release from specific neuronal types by recombinant tetanus toxin light chain fused to antibody variable region.

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