Neurotrophic effects of Botulinum neurotoxin type A in hippocampal neurons involve activation of Rac1 by the non-catalytic heavy chain (HCC/A)

Abstract

Botulinum neurotoxins (BoNTs) are extremely potent naturally occurring poisons that act by silencing neurotransmission. Intriguingly, in addition to preventing presynaptic vesicle fusion, BoNT serotype A (BoNT/A) can also promote axonal regeneration in preclinical models. Here we report that the non-toxic C-terminal region of the receptor-binding domain of heavy chain BoNT/A (HC_C/A) activates the small GTPase Rac1 and ERK pathway to potentiate axonal outgrowth, dendritic protrusion formation and synaptic vesicle release in hippocampal neurons. These data are consistent with HC_C/A exerting neurotrophic properties, at least in part, independent of any BoNT catalytic activity or toxic effect.

Keywords: Botulinum neurotoxin; ERK; Hippocampal neuron; Neurotrophy; Rac1.

Fig. 1

BoNT/A(0) and HC_C/A activate Rac1 and ERK1–2. A) Schematic representing the domains of BONT(0), including the HC_C/A used here. B) Representative Western blot of samples from DIV14–17 cortical neurons treated with vehicle, 25 nM BoNT/A(0) or 25 nM HC_C/A for 30 min. Activated Rac1 was isolated using Active Rac1 Detection Kit (Cell Signalling Technologies). Isolated active Rac1 and cell lysate (for total Rac1) were then subjected to Western blotting and probed for Rac1. C) Quantification of the results in B, represented by mean values ± SEM. ANOVA followed by Tukey post hoc test. *p < 0.05. N = 3. D) Representative Western blot from DIV14–17 cortical neurons treated with vehicle, 25 nM BoNT/A(0) or 25 nM HC_C/A for 30 min. Membranes were probed with anti-pERK and anti-ERK antibodies. E) Quantification of the results in D, represented by mean values ± SEM. ANOVA followed by Tukey post hoc test. *p < 0.05, * *p < 0.01. N = 4.

Fig. 2

BoNT/A(0) and HC_C/A do not globally enhance neurite outgrowth. A) Representative images of DIV7 hippocampal neurons grown in the absence or presence of 25 nM BoNT/A(0) or HC_C/A with our without the Rac1 inhibitor NSC23766 (scale bar = 100 µM). B) Quantification of relative neurite outgrowth over time, represented as mean ± SEM. ANOVA followed by Tukey post hoc test, * *p < 0.01 at the end of the experiment. N = 4.

Fig. 3

BoNT/A(0) and HC_C/A induce axon outgrowth in a manner that requires Rac1 activity. A) Representative images of DIV2 hippocampal neurons grown with 25 nM BoNT/A(0) or HC_C/A in the absence or presence of 100 µM NSC2736. Axons with positive AnkG staining are shown as red and outlined by dotted lines. Blue signal corresponds to nuclei (DAPI). Scale bar = 10 µm. B) Quantification of axonal outgrowth, represented as mean ± SEM. ANOVA followed by Tukey post hoc test, *p < 0.05, * *p < 0.01. Analysis of at least 3 cells per coverslip using coverslips from 3 independent cultures per condition. N = 3.

Fig. 4

BoNT/A(0) and HC/A increase the number of dendritic protrusions. A) Representative images of DIV14–17 hippocampal neurons transfected with a plasmid encoding eGFP and treated for 3 days with 25 nM BoNT/A(0) or HC_C/A in the absence or presence of 100 µM NSC23766. B) Quantification of the number of protrusions per unit of neurite length, represented as mean values ± SEM. ANOVA followed by Tukey post hoc test. *p < 0.05. Analysis of at least 3 cells per coverslip using coverslips from 3 independent cultures per condition. N = 3. Scale bar = 10 µm.

Fig. 5

HC_C/A promotes release of the reserve pool of synaptic vesicles, A) Representative images of from DIV14–16 hippocampal neurons transfected with SypHy and mCherry. Cells were treated for 3 days with 25 nM BoNT/A(0) or HC_C/A and, after baseline recordings, stimulated to release the readily-releasable pool of synaptic vesicles (66 APs at 33 Hz) followed, 10 s later, by a stimulation of 900 APs at 20 Hz to release the reserve pool. The maximal fluorescence was revealed by NH₄Cl, which temporally equilibrates all intracellular compartments to the extracellular pH 7.4. B) Quantification of the SypHy signal, represented as mean ± SEM. Red arrows indicate stimulations described in A. C) Quantification of reserve pool release showing the average ΔF/F_max comprising values from 10 s after the second stimulation until the peak begins to subside at 70 s. Analysis of at least 3 cells per coverslip using coverslips from 3 independent cultures per condition. * *p < 0.01. N = 3.

Fig. 6

BoNT/A(0), HC_C/A and NSC23766 increase NSC neuronal differentiation. A) Representative images of DIV10 NSCs grown in non-differentiated conditions in the absence or presence of 25 nM BoNT/A(0) or HC/A, with or without 100 µM NSC23766, and stained with anti-Tuj1 (green) as a marker for differentiation and DAPI (blue) to stain nuclei. Scale bar = 10 µM. B) Quantification of the proportion of Tuj1-positive cells represented as mean ± SEM. ANOVA followed by Tukey post hoc test. *p < 0.05, * *p < 0.01. Analysis of multiple fields of view per coverslip using coverslips from 3 independent cultures per condition. N = 3.