Neuritogenic actions of botulinum neurotoxin A on cultured motor neurons

Abstract

Botulinum neurotoxins (BoNTs) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage to inhibit neurotransmitter release. The ability of BoNT serotype A (BoNT/A) to eliminate localized transmitter release at extremely low doses is well characterized. In the current study, we investigated the less understood characteristic of BoNT/A to induce nerve outgrowth, sometimes referred to as sprouting. This phenomenon is generally considered a secondary response to the paralytic actions of BoNT/A, and other potential factors that may initiate this sprouting have not been investigated. Alternatively, we hypothesized that BoNT/A induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) synaptosomal associated protein of 25 kDa (SNAP-25). To test this, the effects of BoNT/A application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord. In this system, BoNT/A potently stimulated neuritogenesis at concentrations as low as 0.01 nM. The neuritogenic effects of BoNT/A exposure were concentration dependent and antagonized by Triticum vulgaris lectin, a known competitive antagonist of BoNT. Similar results were observed with the isolated BoNT/A binding domain, revealing that neuritogenesis could be initiated solely by the binding actions of BoNT/A. In addition, the presence or absence of SNAP-25 cleavage by BoNT/A was not a determinant factor in BoNT/A-induced neuritogenesis. Collectively, these results suggest that binding of BoNT/A to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s), independent of its known action on vesicular release.

Fig. 1.

Neurite outgrowth of cultured eMNs. Comparison of a single dose of BoNT/A at three time points. A, fluorescent images of individual eMNs taken from cultures treated with 0.1 nM BoNT/A (BoNT) or without (C) for 24, 48, and 72 h. Neurons were imaged using an antibody to protein gene product 9.5. Scale bar, 50 μm. B, comparisons of total neurite length in μm (mean ± S.E.M.) between control (○, n = 30) and BoNT/A (□, n = 30)-treated neurons at 24, 48, and 72 h. ***, value is significantly different from control at p < 0.001. Inset, phase-contrast images of neurons at 4 h postplating just before treatment.

Fig. 2.

Concentration-dependent effects of BoNT/A on three different parameters of neurite outgrowth. A, fluorescent images of eMNs taken from cultures treated with increasing concentrations of BoNT/A (0–10 nM) at 24 and 48 h. Neurons imaged using an antibody to protein gene product 9.5. Scale bar, 50 μm. B, comparisons of total neurite length in micrometers (mean ± S.E.M., n = 30) from the lowest (0 nM) to the highest (10 nM) concentrations at 24 and 48 h. Exception: n = 28 for 48-h 0.01 nM group. C and D, comparisons of the numbers of primary (C) and secondary (D) neurites from 0.01 to 10 nM BoNT/A at 24 and 48 h. Data are represented as the mean percentage change from control (0 nM) values ± S.E.M., n = 30. B to D, ***, **, and *, values are significantly different from control (0 nM) at p < 0.001, p < 0.01, and p < 0.05, respectively; ‡, value is significantly different from all other concentrations at p < 0.001.

Fig. 3.

SNAP-25 protein in eMNs. Top, immunoblot of SNAP-25 protein isolated from eMN cultures at 4.5, 5, and 6 h after plating. Bottom, immunoblots of eMN cultures treated with either 0.1 nM BoNT/A holotoxin (left) or 0.1 nM BoNT/A H_C (right) at 24 and 48 h. The presence of two bands in blots from holotoxin-treated cultures indicates toxin-induced cleavage, with the lower band representing cleaved SNAP-25. Note the absence of the cleaved SNAP-25 bands in the H_C-treated cultures. Protein samples (10 μg/lane) were resolved on 12.5% Tris-HCl gels. SNAP-25 immunoreactivity was detected with rabbit SNAP-25 primary antibody and visualized by chemiluminescence.

Fig. 4.

Concentration-dependent effects of BoNT/A binding domain (H_C) on neurite outgrowth. A, comparisons of total neurite length in micrometers (mean ± S.E.M., n = 30) from the lowest (0 nM) to the highest (10 nM) concentrations of H_C at 24 and 48 h. B and C, comparisons of the numbers of primary (B) and secondary (C) neurites from 0.01 to 10 nM H_C at 24 and 48 h. Data are represented as the mean percentage change from control (0 nM) values ± S.E.M., n = 30. A to C, ***, **, and *, values are significantly different from control (0 nM) at p < 0.001, p < 0.01, and p < 0.05, respectively.

Fig. 5.

Antagonism of BoNT/A holotoxin (A) and H_C (B) effects on total neurite length by ganglioside blockade using TVL. For each group, data are presented as mean ± S.E.M. (n = 30). TVL used at 1 μM. *** and *, values are significantly different from controls at p < 0.001, and p < 0.05, respectively.