The Evaluation of the Potential of Botulinum C3 Enzyme as an Exogenous Differentiation Inducing Factor to Neurons.

Abstract

ABSTRACT. Botulinum C3 enzyme produced by Clostridium botulinum type C and D strains modifies Rho proteins. In a previous study, we observed that the LDH isozyme pattern of neurons treated with C3 enzyme was different from that induced with endogenous growth factor of neurons such as NGF [21]. This type of change is considered to have an advantage in the medical use of C3 enzyme for neural disorder. To determine the functional similarity of C3-treated neurons to control and NGF-treated neurons, we examined the responses of C3-treated neurons to various drugs, including some neurotransmitters, by measuring the rise of intracellular Ca ions into the neurons. The time course of the rise of intracellular Ca ions induced by high concentration of potassium in the C3-treated neurons was similar to that in the NGF-treated neurons. The C3-treated neurons responded to glutamic acid, aspartic acid, kainic acid, γ-aminobutylic acid, muscarine and ACh with similar time courses and magnitudes as the control neurons. These results suggest that the C3 enzyme induces the functional differentiation of neurons, and that C3 enzyme has the potential for the medical use as an exogenous differentiation-inducing factor of neurons.-KEY WORDS: botulinum C3 enzyme, Ca 2+ , differentiation, neuron, neurotransmitter.Botulinum C3 enzyme was first reported as a new type of botulinum toxin with an ADP-ribosyl transferase activity [1]. C3 enzyme has the unique function of inducing a morphological change in cells when they are incubated with it. Since the morphological change of neural cells caused by C3 enzyme seems to be a differentiation of cells, the effects of C3 enzyme on induction of differentiation marker enzymes have been examined. C3 enzyme increases choline acetyl transferase, acetylcholine esterase and 2', 3'-cyclic-nucleotide 3'-phosphohydrolase in NG108-15 and C6 cell lines [12]. These facts suggest that C3 enzyme has a differentiation-inducing activity on neurons.We obtained results in previous studies on the change of the lactate dehydrogenase (LDH) isozyme pattern; the ratio of H-subunit to M-subunit in LDH was decreased significantly by C3 enzyme treatment in primary cultured neurons. Also, the ratio of H/M in LDH was increased by nerve growth factor (NGF) and N6, 2'-O-dibutyryl 3', 5'-cyclic monophosphate (DBcAMP) [21]. These results suggest that the C3 enzyme induces an intracellular state in which less oxygen is consumed in primary cultured neurons. The change in the characteristics of the neuron to the intracellular reduced oxygen consumption state has important benefits for the treatment of ischemia. These results motivated us to study the potential of the C3 enzyme as an exogenous differentiation-inducing factor for neurons. To evaluate the potential of C3 enzyme, it is necessary to determine the response of the C3-treated neurons to various physiological and pharmacological substances.In this study, we examined the responses of C3-treated neurons to various drugs, including some neurotransmitters, by measuring the rise o...

C3 exoenzyme fromClostridium botulinum: structure of a tetragonal crystal form and a reassessment of NAD-induced flexure

“…This process brings about a variety of cellular changes, some of which are morphological: adherent cells`round up' (Chardin et al, 1989), monocytes spread out (Aepfelbacher et al, 1996) and neuronal PC12 cells exhibit neurite outgrowth (Rubin et al, 1988). C3bot1 also stimulates the regeneration of injured axons in vivo (Lehmann et al, 1999), highlighting its potential for the treatment of certain neural disorders (Watanabe et al, 2000). However, the role of C3 exoenzymes in the pathogenesis of bacterial disease is unclear because of the lack of an obvious cell-entry mechanism.…”

Molecular recognition of an ADP-ribosylating Clostridium botulinum C3 exoenzyme by RalA GTPase

“…These functions of Rho are sufficient to explain the distinctive morphological changes observed after the addition of C3bot1 to the cytosol of cells. These changes are specific to cell type, and the observation that neuronal PC12 cells exhibit neural outgrowth (12) and experiments showing that C3bot1 can cause regeneration of injured axons in vivo (13) suggested a possible usefulness in treating neuronal disorders (14).…”

Characterization of a Neutralizing Monoclonal Antibody against Botulinum ADP-Ribosyltransferase, C3 Exoenzyme.

“…C3 exoenzyme evoked the outgrowth of neurites from a chicken ganglion [7] and induced acetylcholinesterase activity in PC12 cells [15], NG108-15, and C6 cell lines [8]. The primary culture cells of mouse cerebrum treated with C3 exoenzyme showed a response similar to that of the control matured cells against various drugs such as muscarine, and acetylcholine as a neurotransmitter [21]. These observations then indicate that C3 exoenzyme is regarded as a kind of neurotropic factor.…”