Heat map comparison of significantly regulated genes after 2 weeks of exposure to BoNT/A1 and BoNT/A <i>ad</i>.

<p>All genes differentially expressed greater than 2-fold in either BoNT/A1 or BoNT/A <i>ad</i>-exposed cells are represented, sorted by normalized expression level as determined by the first replicate of BoNT/A1-exposed cells. All three replicates of each condition are shown as individual columns. Blue represents lower expression, while red represents higher expression on a scale of −3.0 to 3.0.</p

List of all primer sequences used for qPCR.

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

Analysis of Gene Expression in Induced Pluripotent Stem Cell-Derived Human Neurons Exposed to Botulinum Neurotoxin A Subtype 1 and a Type A Atoxic Derivative

<div><p>Botulinum neurotoxin type A1 (BoNT/A1) is a potent protein toxin responsible for the potentially fatal human illness botulism. Notwithstanding, the long-lasting flaccid muscle paralysis caused by BoNT/A has led to its utility as a powerful and versatile bio-pharmaceutical. The flaccid paralysis is due to specific cleavage of neuronal SNAREs by BoNTs. However, actions of BoNTs on intoxicated neurons besides the cleavage of SNAREs have not been studied in detail. In this study we investigated by microarray analysis the effects of BoNT/A and a catalytically inactive derivative (BoNT/A <i>ad</i>) on the transcriptome of human induced pluripotent stem cell (hiPSC)-derived neurons at 2 days and 2 weeks after exposure. While there were only minor changes in expression levels at 2 days post exposure, at 2 weeks post exposure 492 genes were differentially expressed more than 2-fold in BoNT/A1-exposed cells when compared to non-exposed populations, and 682 genes were differentially expressed in BoNT/A <i>ad</i>-exposed cells. The vast majority of genes were similarly regulated in BoNT/A1 and BoNT/A ad-exposed neurons, and the few genes differentially regulated between BoNT/A1 and BoNT/A <i>ad</i>-exposed neurons were differentially expressed less than 3.5 fold. These data indicate a similar response of neurons to BoNT/A1 and BoNT/A <i>ad</i> exposure. The most highly regulated genes in cells exposed to either BoNT/A1 or BoNT/A <i>ad</i> are involved in neurite outgrowth and calcium channel sensitization.</p></div

List of genes differentially regulated greater than 2-fold at 2 days post toxin exposure.

<p>List of genes differentially regulated greater than 2-fold at 2 days post toxin exposure.</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 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