Botulinum neurotoxins A, B, C, E, and F preferentially enter cultured human motor neurons compared to other cultured human neuronal populations

Abstract

Human-induced pluripotent stem cell (hiPSC)-derived neurons can be exquisitely sensitive to botulinum neurotoxins (BoNTs), exceeding sensitivity of the traditionally used mouse bioassay. In this report, four defined hiPSC-derived neuronal populations including primarily GABAergic, glutamatergic, dopaminergic, and motor neurons were examined for BoNT/A, B, C, D, E, and F sensitivity. The data indicate that sensitivity varies markedly for the BoNTs tested. Motor neurons are significantly more sensitive than other neuron types for all BoNTs except BoNT/D. Examination of SNARE protein levels and BoNT-specific cell surface protein receptors reveals few differences between the cell types except greater expression levels of the receptor protein SV2C and synapsin-IIa in motor neurons. This indicates that differential toxicity of BoNTs for motor neurons compared to other neuronal cell types involves multiple mechanisms.

Keywords: botulinum neurotoxin; cell-based assay; hiPSC; human cell lines; neurons.

Figure 1

Human induced pluripotent stem cell (hiPSC)-derived neuronal cell models used in this study at day 14 in culture. Cells were stained with CellTracker Green CMFDA, and images were obained by fluorescent microscopy using an EVOS Aufto FL2 scope with the GFP filter. The size-bar is 75 µm.

Figure 2

SNARE cleavage Western blots showing cell type and BoNT type specificity. Neuronal cell populations were grown for 14 days, and exposed to BoNT/A, /B, /E, or /F for 48 h in parallel. Cell lysates were analyzed for SNARE cleavage by Western blot using an anti SNAP-25 antibody that recognizes both BoNT/A or /E cleaved and uncleaved SNAP-25 and an anti-VAMP2 antibody which shows only intact uncleaved VAMP2, with syntaxin used as a loading control. All samples were tested in triplicate and one representative Western blot of each is shown. The type of neurons is indicated by name supplied by the manufacturer (Fujifilm CDI).

Figure 3

SNARE protein and BoNT protein receptor protein levels in the four hiPSC-derived cell models. Neuronal cell populations were grown for 14 days, and cell lysates analyzed for the indicated proteins by Western blot. Beta-actin was used as a loading control. The left panel shows a representative blot of triplicate samples. The graph shows a relative depiction of average and standard deviations of the triplicate samples comparing expression levels of each protein between the four cell populations. Protein bands were quantified by densitometry relative to the beta-actin loading control. Protein levels were then set to 100 % for motor-neurons, except for SV2A and B, which were detected only at very low levels in motor-neurons and were adjusted to 100 % in GABA Neurons.

Figure 4

Western blots showing VAMP1 and VAMP2 cleavage by BoNT/D in human Motor Neurons. Motor Neurons were grown for 14 days, and exposed to BoNT/D for 48 h. The same cell lysates were analyzed for VAMP1 and VAMP2 cleavage by Western blot. Syntaxin was used as a loading standard. All samples were tested in triplicate and one representative Western blot of each is shown.