Sequential uptake of BoNTs.

<p>Human iPSC derived neurons (iCell Neurons) (a, b, c) or primary mouse spinal cord cells (d) were exposed to the indicated BoNT serotypes (10 U / well BoNT/A, 1000 U / well BoNT/B, 1,000 U / well BoNT/D, 2,000 U / well BoNT/E, and 30 U / well BoNT/F) for 48 h to cause full SNARE cleavage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133737#pone.0133737.s001" target="_blank">S1 Fig</a>). After complete removal of all extracellular toxin, the pre-exposed and not pre-exposed neurons were exposed to serial dilutions of the indicated BoNTs for 48 h in parallel, and cell lysates were analyzed for SNARE cleavage by Western blot using an anti-SNAP-25 antibody that recognizes both cleaved and uncleaved SNAP-25 equally and a VAMP2 antibody that detects only uncleaved VAMP2, which is quantitated in relation to syntaxin (all antibodies from Synaptic Systems). The graphs show the quantitative analyses of SNARE cleavage by the second BoNT serotype (as shown on the x-axis label), and the graph legends indicate the BoNT serotype added for pre-exposure. C: Cells first exposed to BoNT/E followed by exposure to BoNT/A were incubated for an additional 3 weeks before cell lysis to allow for recovery of BoNT/E cleaved SNAP-25. In all cases, each data point was measured in triplicate, and data were analyzed for statistical significance using an F-test in PRISM 6 software (n = 3).</p

Botulinum Neurotoxins Can Enter Cultured Neurons Independent of Synaptic Vesicle Recycling

<div><p>Botulinum neurotoxins (BoNTs) are the causative agent of the severe and long-lasting disease botulism. At least seven different serotypes of BoNTs (denoted A-G) have been described. All BoNTs enter human or animal neuronal cells via receptor mediated endocytosis and cleave cytosolic SNARE proteins, resulting in a block of synaptic vesicle exocytosis, leading to the flaccid paralysis characteristic of botulism. Previous data have indicated that once a neuronal cell has been intoxicated by a BoNT, further entry of the same or other BoNTs is prevented due to disruption of synaptic vesicle recycling. However, it has also been shown that cultured neurons exposed to BoNT/A are still capable of taking up BoNT/E. In this report we show that in general BoNTs can enter cultured human or mouse neuronal cells that have previously been intoxicated with another BoNT serotype. Quantitative analysis of cell entry by assessing SNARE cleavage revealed none or only a minor difference in the efficiency of uptake of BoNTs into previously intoxicated neurons. Examination of the endocytic entry pathway by specific endocytosis inhibitors indicated that BoNTs are taken up by clathrin coated pits in both non pre-exposed and pre-exposed neurons. LDH release assays indicated that hiPSC derived neurons exposed consecutively to two different BoNT serotypes remained viable and healthy except in the case of BoNT/E or combinations of BoNT/E with BoNT/B, /D, or /F. Overall, our data indicate that previous intoxication of neuronal cells with BoNT does not inhibit further uptake of BoNTs.</p></div

Botulinum Neurotoxins B and E Translocate at Different Rates and Exhibit Divergent Responses to GT1b and Low pH

Botulinum neurotoxins (BoNTs, serotypes A–G) are the most deadly substances known. Here, we investigated how BoNT/E, a serotype that causes human botulism, translocates into the cytosol of neurons. Analogous to BoNT/B, BoNT/E required binding of the coreceptor, GT1b, to undergo significant secondary structural changes and transform into a hydrophobic protein at low pH. These data indicate that both serotypes act as coincidence detectors for both GT1b and low pH, to undergo translocation. However, BoNT/E translocated much more rapidly than BoNT/B. Also, BoNT/E required only GT1b, and not low pH, to oligomerize, whereas BoNT/B required both. In further contrast to the case of BoNT/B, low pH alone altered the secondary structure of BoNT/E to some degree and resulted in its premature inactivation. Hence, comparison of two BoNT serotypes revealed that these agents exhibit both convergent and divergent responses to receptor interactions, and pH, in the translocation pathway

Cytotoxicity of sequential exposure of human neurons to different BoNT serotypes.

<p>Human iPSC derived neurons (iCell Neurons) were exposed to 5 nM BoNT/A or BoNT/E for 24 h, followed by sequential exposure to 5 nM BoNT/B, /D, or /F for 24 h. Culture media was examined at 0–24 h, 24–48 h, and 48–72 h post addition of the second toxin for LDH release indicating cytotoxicity. Lysis buffer for the 100% lysed cells was added at 0 h (for the 0–24 h time point), 24 h (for the 24–48 h time point), and 48 h (for the 48–72 h time point). Media was replaced between each time point. All samples were measured in triplicate, and the average and standard deviation are shown in the graph. Statistical significance in relation to the control (cells with no toxin added) was determined by students t-test. Values that were significantly different at all three time points are indicated by a * (p < 0.01). One set of cells was lysed at 24 h post addition of the second toxin, and cell lysates were analyzed for SNARE cleavage by Western blot. One representative Western blot of triplicate samples is shown.</p

Activity dependent sequential BoNT entry into neurons.

<p>Neuronal cultures were pre-exposed to BoNT/B or BoNT/F for 48 h, followed by complete removal of extracellular toxin. The pre-exposed or not pre-exposed cells were then exposed to the indicated concentrations (U / well) of BoNT/A1 in culture media or cell stimulation media (containing 56 mM KCl and 2.2. mM CaCl2) for 5 min, followed by removal of toxin, washing of the cells, and incubation for 24 h to allow for SNAP-25 cleavage. Cell lysates were analyzed for SNARE cleavage by Western blot and densitometry. All samples were tested in triplicate. (a) Human iPSC derived neurons (iCell Neurons) pre-exposed to 30 U BoNT/F. A Representative Western blot of SNAP-25 cleavage in depolarized (cell stimulation media) or not depolarized (culture media) cells is shown in the top figure, and VAMP-2 cleavage in BoNT/F pre-treated neurons in shown in the bottom figure. (b) Primary mouse spinal cord cells (top) or iCell Neurons (bottom) were exposed to 20,000 U BoNT/B / well (MSC cells) or 1000 U BoNT/B / well (iCell Neurons) for 48 h to cleave all VAMP2, followed by exposure to the indicated concentrations (U / well) of BoNT/A1 in culture media or cell stimulation media (containing 56 mM KCl and 2.2. mM CaCl2) for 5 min. Toxin was removed, cells washed and incubation for 24 hgig. to allow for SNAP-25 cleavage. Representative Western blots are shown. (c) Human iPSC derived neurons (iCell Neurons) pre-exposed to 1000 U BoNT/B for 48 h (bottom image), and after toxin removal cells were depolarized with 5 sequentially rounds of depolarization in cell stimulation media before exposure to serial dilutions of BoNT/A1 in either cells stimulation media for 5 min, or in culture media for 24 h. The graphs depict quantitative data from triplicate samples, respectively, and statistical significance was evaluated using an F-test in PRISM6.</p

Inhibition of BoNT/A entry into human neurons by endocytosis inhibitors specific for clathrin dependent endocytosis.

<p>(a) Human iPSC derived neurons (iCell Neurons) were treated with the indicated endocytosis inhibitors for 30 min at 37°C. The cells were then exposed to 200 U of BoNT/A1 / well for 7 h, and cell lysates analyzed for SNAP-25 cleavage by Western blot. B: Human iPSC derived neurons (iCell Neurons) were exposed to 1000 U / well of BoNT/B to achieve full VAMP2 cleavage (top panel). Toxin was removed, and cells were treated with the indicated endocytosis inhibitors for 30 min at 37°C. The cells were then exposed to 200 U of BoNT/A1 / well for 5 min in either culture media or cell stimulation media, followed by toxin removal and further incubation for 8 h. Cell lysates were analyzed for SNAP-25 cleavage by Western blot. SNAP-25 cleavage in depolarized (cell stimulation media) or not depolarized (culture media) cells is shown in the Western blot, and the respective inhibitors are indicated in the table.</p

Number of genes differentially regulated between BoNT/A1 and BoNT/A <i>ad</i>-exposed cells at 14 days post toxin exposure.

<p>When two differentially regulated entities were identified as the same gene, the average of the differential expression was used for categorization.</p><p>Number of genes differentially regulated between BoNT/A1 and BoNT/A <i>ad</i>-exposed cells at 14 days post toxin exposure.</p

List of select genes differentially regulated at 2 weeks post-exposure, with direction in relation to non-exposed populations.

a<p>The first number represents regulation of BoNT/A1 in relation to non-exposed samples, the second number represents regulation of BoNT/A <i>ad</i> in relation to non-exposed samples.</p><p>List of select genes differentially regulated at 2 weeks post-exposure, with direction in relation to non-exposed populations.</p

Confirmation of BoNT activity in exposed and non-exposed iCell Neuron samples by Western blot.

<p>A representative Western blot is shown for both 2 days and 14 days exposure. Percentage of SNAP-25 cleavage, conducted via densitometry and averaged for all three replicates after correcting for background values, is represented as a bar graph; error bars represent standard deviation of the three replicates.</p

List of all primer sequences used for qPCR.

<p>List of all primer sequences used for qPCR.</p