Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinumToxin A Endopeptidase Fragment and Erythrina cristagalliLectin

Abstract

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH N /A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH N /A or recombinant LH N /A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH N /A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved. The clostridial neurotoxin (CNT)1 family includes tetanus toxin (TeNT), produced by Clostridium tetani, and the seven antigenically distinct botulinum neurotoxins produced from strains of Clostridium botulinum (BoNTs). These proteins are responsible for the conditions of tetanus and botulism, respectively, that develop as a direct result of inhibition of Ca 2ϩ -dependent neurotransmitter release, a mechanism of action common to all the CNTs. In the case of BoNTs, intoxication of the neuromuscular junction is thought to occur in at least three phases: an initial binding phase, an internalization phase, and finally a neurotransmitter blockade phase (1).All CNTs have a similar structure and consist of a heavy chain (ϳ100 kDa) covalently joined to a light chain (ϳ50 kDa) by a single disulfide bond. Proteolytic cleavage of the heavy chain of C. botulinum neurotoxin type A (BoNT/A) generates two fragments of ϳ50 kDa each. The C-terminal domain (H C ) is required for target cell binding, with the N-terminal domain (H N ) being proposed to be involved in intracellular membrane translocation (2). Under conditions in which the disulfide bond between the light and heavy chains is maintained, trypsin cleavage results in a 100-kDa species termed LH N /A (light chain plus N-terminal heavy chain domain) representing a catalytically active, non-cell binding, non-toxic derivative of BoNT/A. In addition to obtainin...

Botulinum A Toxin Intravesical Injections in the Treatment of Painful Bladder Syndrome: A Pilot Study

“…Recently, the ability of a catalytically active derivative of BTX-A to block the secretion from vesicle populations in dorsal root ganglia neuronal cultures has been demonstrated for the release of substance P and glutamate, which are involved in afferent nociceptive transmission to the central nervous system [8]. This evidence supports the clinical application of BTX-A in the treatment of bladder hypersensitive disorders in humans.…”

Botulinum-A Toxin Injections Into the Detrusor Muscle Decrease Nerve Growth Factor Bladder Tissue Levels in Patients With Neurogenic Detrusor Overactivity

“…15 These axons release inflammatory mediators, such as substance P, neurokinin A and CGRP, into peripheral tissues, which exert direct vasoactive effects (vasodilatation and increased vasopermeability) with consequent tissue edema. The block of secretion from vesicle populations in dorsal root ganglia neuronal cultures of substance P and glutamate, as induced by BTX-A, 3 can decrease the effects of neurogenic inflammation. Furthermore, during followup patients did not show any urinary tract infection and the majority did not report any infection at a mean followup of 6 months.…”

“…Transmission of this signal depends on the release of a number of transmitters, including glutamate, substance P and CGRP, from synaptic vesicles. 3 Duggan et al noted that a conjugate obtained by the coupling of a derivative of BTX-A with a lectin shows in vitro selectivity for nociceptive afferents, resulting in the inhibition of neurotransmitters from synaptic vesicles of dorsal root ganglia neuronal cultures. 3 In addition, it was recently reported that intravesical BTX-A significantly decreases afferent nerve mediated contractions to electrical and chemical stimulation in treated rats compared to those in saline treated animals.…”

“…1 An observation is that the neurotoxin inhibits the release of neurotransmitters from the terminals of primary nociceptive afferents by its proteolytic light chain, which hydrolyzes key components of the SNARE (SNAP receptor) complex required for synaptic vesicle docking, fusion and neurotransmitter release. 3 Recently the ability of a catalytically active derivative of BTX-A to block secretion from vesicle populations in dorsal root ganglia neuronal cultures was noted for the release of substance P and glutamate, which are involved in afferent nociceptive transmission to the central nervous system. 3 Experimental studies indicate that detrusor overactivity after SCI is induced at least in part by phenotypic changes in unmyelinated C-fiber bladder afferent pathways.…”

“…3 Recently the ability of a catalytically active derivative of BTX-A to block secretion from vesicle populations in dorsal root ganglia neuronal cultures was noted for the release of substance P and glutamate, which are involved in afferent nociceptive transmission to the central nervous system. 3 Experimental studies indicate that detrusor overactivity after SCI is induced at least in part by phenotypic changes in unmyelinated C-fiber bladder afferent pathways. 4 The hyperexcitability of C-fiber bladder afferents may be mediated at least in part by an increase in NGF, which is produced in target organ tissues and transported retrograde back to the neuronal cell bodies.…”