Differentiated BoNT/A reporter cell line, Red SNAPR, is highly sensitive to BoNT/A intoxication.

(A) Schematic diagram of the BoNT/A reporter construct, SNAPR. Representative images of ReNcell VM cell line, Red SNAPR, stably expressing SNAPR and incubated with 100nM BoNT/A for 48 hours. (B) Western blot of cell lysates from (A) probed with tRFPT and tGFP antibodies. (C) Schematic diagram of the BoNT/A intoxication assay. (D) Red SNAPR differentiated in normal or ReDS medium, then incubated with 0, 10 and 100 nM BoNT/A for 48 hours. Quantification of EC50 dose response of BoNT/A in ReD SNAPR cells incubated with 0 - 100nM BoNT/A in normal and ReDS medium using GFP/RFP ratio readout. (E) Western blot of cell lysates from (D) probed with tRFPT and tGFP antibodies. Scale bars 50 μm.

Differentiation and siRNA depletion dynamics in ReNcell VM.

(A) ReNcell VM differentiated for 2 weeks with staining for nuclei (in blue) and neuronal markers b3-tubulin MAP2 (green) and for oligodendrocyte marker CNPase (2’,3’-Cyclic-Nucleotide3’-Phosphodiesterase). Scale bars 20 μm. (B) ReD SNAPR cell line differentiated for 2 weeks with staining of b3-tubulin (white). Expression of SNAPR construct is detected in the RFP and GFP channels. Scale bars 50 μm. (C) Undifferentiated and differentiated ReNcell VM transfected with siNT3 (Non Targeting 3) siRNA or siRNA against thioredoxin reductase 1 (TXNRD1) for 3 and 5 days showing persistent TXNRD1 protein depletion. Scale bars 50 μm. (D) Undifferentiated and differentiated ReD SNAPR cells transfected with siNT3 or SNAP25 siRNA for 3 and 5 days showing reduction of the SNAPR reporter. Scale bars 20 μm.

BoNT is first detected in the neuronal soma then emanate to the axons.

(A) Red SNAPR cells co-cultivated at ¼ with ¾ of unlabeled Ren-VM were imaged for 48h after addition of BoNT/A. The GFP color-coded intensity signal is displayed in the second column. (B) Quantification of GFP/RFP signal along the length of individual neurites (Axon 1,2,3) at various time points. (C) Schematic diagram of split-mNG (NeonGreen Fluorescent Protein) detection system, consisting of ReNcell VM expressing cytosolic mNG1-10 and mNG11-tagged BoNT/A. Fluorescence occurs after binding of mNG11-tagged BoNT/A to mNG1-10. (D) Time-course of fluorescence reconstitution after exposure to mNG11-tagged BoNT/A and quantification of Mean Fluorescence Intensity in the soma and neurites of cells. Scale bars 20 μm.

Genome-wide RNAi screen of BoNT/A-treated Red SNAPR cell line.

(A) Schematic diagram of assay pipeline. (B) Cells treated with non-targeting control siRNA (siNT3) and positive control siRNA (siTXNRD1), then incubated with BoNT/A for 48 hours. Cells were stained with DAPI and TXNRD1 antibody post-fixation. Scale bars 50 μm. (C) ScreenSifter Z-score analysis of GFP/RFP ratio index of whole genome siRNA library plates. (D) R-squared analysis of both genome-wide RNAi screen replicates. Blue dots = No toxin control, Red dots = Toxin control, Green dots = Positive control (siTXNRD1). (E) Descending Z-score of control-normalized and averaged GFP/RFP ratio of genome-wide screen to determine cut-off values of +1.5 for positive regulators and −3 for negative regulators. (F) Chronological order of genome-wide control-normalized screen reflecting the positive regulators (red) and negative regulators (blue) derived from (E).

Genome-wide RNAi screen data normalization and refinement.

(A) Genome-wide siRNA library plate replicates (Exp1 and Exp2) from Figure 2C (B) Individual replicates from Figure 2D. (C) Averaged nuclei count for each siRNA, 289 genes result in cell number below the cut-off of 4000 nuclei per well. Gene targets of siRNA most affecting cell survival are annotated in the graph. (D) The 289 genes from C are over-imposed on the SNAPR reporter results, 80 genes fall within the positive regulators and 8 genes with the negative regulators. All 88 genes were excluded from further analysis (E) Targeted siRNA deconvolution screen with positive and negative hits and siRNA control either non-toxin treated (NT3-) or toxin-treated (NT3+). Positive controls (TXNRD1+) replicates. The GFP/RFP threshold was set at 1.9 for positive regulators and 1.0 for negative regulators. Green data points are individual siRNAs that do not pass the threshold and do not confirm the pool result. Averaged GFP/RFP ratio for genes below or above threshold lines for positive and negative regulators that were removed from the hit lists. (F) RNA sequencing data from ReD SNAPR cells represented as log2CPM (counts per million) with an exclusion threshold set at CPM = 0.25 (log2CPM = −2, genes below are deemed as unexpressed).

Surface expression of the BoNT/A receptor SV2 is regulated by endocytic trafficking and signaling genes, forming a cohort of the positive regulators of the genome-wide screen.

(A) Detecting surface SV2 using a specific antibody against the extracellular loop of SV2. (B) siVAMP2 as a positive control for surface SV2 expression. ReNcell VM treated with siNT3, siVAMP2 and siTXNRD1 for 3 days and stained with SV2A antibody after fixation without membrane permeabilization. Quantification of surface SV2 per cell where whole image SV2A MFI was divided by total nuclei count (DAPI) from 3 fields of view and 3 independent replicates. Scale bars 50 μm. (C) Replicate screen results for surface SV2 regulators from genome-wide positive hits. Purple box = surface SV2 repressors e.g. siCTLC (clathrin light chain); Brown box = surface SV2 enhancers e.g. siRab11b. siNT3 and siTXNRD1 does not regulate surface levels of SV2. Scale bars 50 μm. (D) STRINGS analysis of surface SV2 repressors. (E) STRINGS analysis of surface SV2 enhancers.

Protein network analysis of genome-wide hits reveals known and novel regulators of BoNT/A intoxication.

(A) Summary diagram of all positive and negative hits mapped to their intracellular localities. (B) STRINGS network analysis of connected hits (non-connected hits are excluded). Associated genes tied to respective cellular molecular complex/processes are bounded and annotated. ScreenSifter analysis revealed (C) Src-associated genes (D) TXNRD1-associated genes (E) Translocon-associated genes (F) Retromer-associated genes.

Gene ontological analysis of positive hits from genome-wide intoxication screen.

(A) Cellular component (B) Molecular function (C) Biological Processes

Retromer is required for retro-axonal trafficking of the BoNT/A receptor.

(A) Cytosolic mNG1-10 expressing RenVM cells were incubated with 50 nM of BoNT/A-mNG11 and fixed at 0, 24, 36 and 48 hour timepoints. The MFI was quantified in 20 cells for each condition and time point, in the soma and neurites. Scale bar 20 μm. (B) ReNcell VM stably expressing Dendra-SV2 treated with siVPS35 for 3 days and imaged after fixation. Insets show axons of interest from each condition and arrowheads indicate enlarged SV2 puncta. Scale bar 20 μm and inset shown at 10 μm. (C) Method for quantifying number and size of SV2 puncta. tGFP image is background-subtracted, thresholded then analyzed for number and size of particles. Quantification of the number of SV2 puncta along every 50 μm segment of the axon starting from the axon tip from B. Quantification of area of SV2 puncta in whole axons from B. (D) Comparison of mean fluorescence intensities (MFI) at axons and axon tips from control and siVPS35-treated cells. Quantification of axon tip/axon MFIs. Graphs are obtained from 30 axons across 3 independent experiments. Scale bar 10 μm.

BoNT/A is retrogradely trafficked through Golgi and ER membranes as revealed by split-GFP reconstitution. (A)

Split-mNG constructs targeted to cytosol, Golgi, ER lumen (KDEL sequence) and ER membranes (Sec61 transmembrane domain) were stably expressed in ReNcell VM and incubated with 50 nM of BoNT/A-mNG11. Control cells (without BoNT/A-mNG11) are shown at top panels of each condition (B) Cells expressing Golgi-mNG1-10 and showing reconstituted mNG fluorescence. Overlap of HA antibody and mNG signals in the Golgi (C) ER-mNG1-10 expressing cells showing mNG fluorescence and distinct overlap of HA antibody and mNG in the ER. (D) Sec61G-mNG1-10 expressing cells showing mNG fluorescence distinct overlap of HA antibody and Sec61G signals. (E) Quantification of MFI/cell from B-D showing increased mNG fluorescence in Golgi, ER and Sec61G-expressing cells after addition of BoNT/A-mNG11. (F) Representative image of SEC61G depletion in ReNcell VM. (G) Golgi-mNG1-10, ER-mNG1-10 and Cytosol-mNG1-10-expressing cells are depleted with siSEC61G and incubated with BoNT/A-mNG11 for 48 hours. (H) Quantification of MFI/cell from G. Graphs are obtained from at least 300 cells across 3 independent experiments. Scale bars 20 μm.

Schematic model for intracellular trafficking of BoNT/A.

(A) BoNT/A HC binds to its cognate receptor SV2. Surface expression of SV2 is regulated by a cohort of enhancers and repressors. (B) BoNT/A-containing endosomes are retro-axonally trafficked to the soma through the retrograde function of the retromer complex. (C) BoNT/A eventually traffics to the translocation-competent ER via the Golgi. (D) The ER SEC61 translocon complex facilitates LC translocation of BoNT/A from the ER lumen into the cytosol where the TXNRD1 and HSP complexes release and refold BoNT/A LC. The LC diffuses and cleaves SNAP25 in the soma. (E) Progressive diffusion of LC into the axons and axon terminals ultimately cleaves SNAP25 on neurotransmitter-containing vesicles, blocking neurotransmission at the synapse.