Article Figures & Data
Figures
- Figure 1.
Axonal retrograde trafficking of BoNT/A-Hc-positive carriers is increased by activity. A, Photograph of the microfluidic devices used to compartmentalize nerve terminals from the somatodendritic area. Scale bar, 1 cm. B, Scheme illustrating the BoNT/A-Hc retrograde transport assay; red spots in the terminal chamber represent BoNT/A-Hc (100 nm). Hippocampal neurons cultured in microfluidic devices were pulse labeled by incubating BoNT/A-Hc-Atto647 (100 nm) for 5 min in the nerve terminal chamber in the presence of low or high K+ buffer, washed in culture medium, and imaged by time-lapse confocal microscopy from 2 to 4 h after the pulse. C, Color-coded arrowheads specify different retrograde carriers. Note that more retrograde carriers are detected in the axons of stimulated neurons. Scale bar, 10 μm. D, E, BoNT/A-Hc-positive carriers were tracked and their frequency (D) and average speed (E) were calculated in the indicated experimental conditions. Data from 5 independent neuron preparations are shown; n = 34 and 31 individual channels for low K+ and high K+, respectively. ***p < 0.001; n.s., not significant, Student's t test.
- Figure 2.
The kinetics of retrograde BoNT/A-Hc carriers is not affected by presynaptic activity. A, Representative kymographs of BoNT/A-Hc carriers generated by a line scan following a single axon track on the time-lapse image stacks. Arrowheads indicate retrograde carriers and asterisks stationary compartments. Scale bars: x-axis, 10 μm; y-axis, 10 s. B, C, Frequency distribution of average speed (B) and instant speed (C) of BoNT/A-Hc carriers after a 5 min pulse in either low K+ or high K+ treatment followed by a 2–4 h chase. Dwelling time defined by tracks times with an instant speed of 0–0.7 μm/s is quantified in C (blue highlight) and D as percentage dwelling time relative to the total track time shown in the indicated conditions. Data from 5 independent neuron preparations are shown; n = 34 and 31 individual channels for low K+ and high K+, respectively. *p < 0.05; n.s., not significant, Student's t test.
- Figure 3.
BoNT/A-Hc single molecules also undergo fast retrograde axonal transport. A, Scheme illustrating BoNT/A-Hc (red spot) axonal retrograde transport in microfluidic devices and the single-molecule detection technique used. Hippocampal neurons grown in microfluidic devices were pulsed 5 min with BoNT/A-Hc-Atto647 (1 nm) added to terminal chamber only. After a 2 h chase, single-molecule detection was performed in the axon channel. B, Selected trajectories of BoNT/A-Hc-Atto647N single molecules tracked at the entrance of the axon channel overlaid with a bright-field image. Shown is a high-power view of a single-molecule trajectory (bottom). Scale bar, 10 μm. C, MSD of the trajectories of all detected BoNT/A-Hc molecules and those selected for undergoing active transport (directed motion). D, Left, Frequency distribution of the average speed of single BoNT/A-Hc molecules (1 nm) and carriers (100 nm). Right, Statistical comparison of the average speeds of BoNT/A-Hc molecules (detected by single-molecule imaging) and fluorescent carriers detected by confocal microscopy. For the 1 nm group, n = 21 axon channels (a total of 176 trajectories from 3 independent neuron preparations). For the 100 nm group, n = 27 axon channels (a total of 571 trajectories from 5 independent neuron preparations). n.s., Not significant, Student's t test.
- Figure 4.
Retrograde BoNT/A holotoxin transport is affected by presynaptic activity. A–D, Both BoNT/A-Hc (Hc/A) uptake (A) and retrograde trafficking (B) were reduced in GFP-Lc/A-transfected neurons. Hc/A intensity of GFP-Lc/A-transfected neurons (box a) compared with Hc/A intensity of adjacent untransfected neuron (box b). SNAP25A was used to verify its cleavage in GFP-Lc/A-transfected hippocampal neurons. Scale bar, 20 μm. C, Quantitative results of A. D, Quantitative results of B. Data from 3 independent cultures are shown; n = 21 and 14 for uptake and retrograde, respectively. **p < 0.01; ***p < 0.001, paired t test. E, Hippocampal neurons grown in microfluidic devices were pulsed for 5 min with 100 pm BoNT/A added to the terminal chamber only, after which the long-range effects of BoNT/A were assessed in unstimulated (low K+) or stimulated (high K+) conditions by immunofluorescence microscopy of SNAP25A. Chambers without BoNT/A addition were used as the negative control (control). Scale bar, 50 μm. F, G, Quantitative results of terminal chamber (F) and soma chamber (G), respectively. Data are from 4 independent cultures; n = 4. *p < 0.05, Student's t test. H, Representative Western blot of soma chamber lysate samples as described above and soma chamber lysate with BoNT/A added directly to the soma chamber as a positive control (lane 1). β-actin was used as the loading control. I, Quantitative results of H, with SNAP25A signal normalized to β-actin. Data are from 3 independent preparations, n = 3. *p < 0.05, paired t test.
- Figure 5.
BoNT/A-Hc carriers partially colocalize with p75NTR and LysoTracker compartments. A–C, Hippocampal neurons cultured in microfluidic devices were incubated with FITC-p75NTR antibody for 1 h at 37°C before exposure to stimulated (high K+) or unstimulated (low K+) conditions, with BoNT/A-Hc added to the terminal chamber only. Neurons were chased for 2–4 h at 37°C and the axon channels were imaged. A, Images of neurons treated with low K+ or high K+. BoNT/A-Hc (Hc/A) carriers colocalized with p75NTR are indicated with arrowheads. Scale bar, 5 μm. B, Kymographs corresponding to the time series described above. Colocalized tracks are indicated with arrowheads. Scale bars: x-axis, 1 μm; y-axis, 10 s. C, Quantification of B. Data are from 3 independent preparations; n = 49 and 46 for low K+ and high K+ respectively. n.s., Not significant, Student's t test. D–F, Hippocampal neurons cultured in microfluidic devices were incubated with LysoTracker Red for 30 min at 37°C before exposure to stimulated (high K+) or unstimulated (low K+) conditions, with BoNT/A-Hc added to the terminal chamber only. Neurons were chased for 2–4 h at 37°C and the axon channels were imaged. D, Images of neurons treated with low K+ or high K+. BoNT/A-Hc (Hc/A) carriers colocalized with LysoTracker are indicated with arrowheads. Scale bar, 5 μm. E, Kymographs corresponding to the time series described above. Colocalized tracks are indicated with arrowheads. Scale bars: x-axis, 1 μm; y-axis, 10 s. F, Quantification of E. Data are from 3 independent preparations; n = 51 and 57 for low K+ and high K+, respectively. **p < 0.01.
- Figure 6.
Electron microscopy of axon channels reveals that BoNT/A-Hc is retrogradely transported by autophagosomes. A, Representative axon bundles from unlabeled neurons with autophagosomes (a1 and a2, boxes) and mitochondria (arrowheads) along the microtubule tracks. Scale bar, 500 nm. B, C, Quantification of autophagosomes (B) and mitochondria (C) observed along the axon bundles in the indicated conditions; n = 3 independent microfluidic devices. *p < 0.05; n.s. not significant, Student's t test. D, Representative electron microscopy of axon bundles of neurons cultured in microfluidic devices with (right) or without (left) BoNT/A-Hc-HRP added to the nerve terminal chambers under high K+ conditions. Autophagosomes are indicated by arrowheads. Scale bar, 500 nm. E, Quantification of BoNT/A-Hc-HRP-containing autophagosome as shown in D; n = 3. **p < 0.01, Student's t test. F, Representative LC3 and βIII-tubulin immunostaining of hippocampal neurons cultured in microfluidic devices and fixed at indicated times after stimulation. Boxed regions are shown in G; arrowheads indicate nerve terminals. H, Quantification of the level of LC3 fluorescence normalized to βIII-tubulin; n = 6 from 2 independent cultures. ***p < 0.001; **p < 0.01, Student's t test. I, Representative LC3 and β-actin (loading control) immunoblots. J, Quantification of I; n = 3. *p < 0.05, Student's t test.
- Figure 7.
Retrograde BoNT/A-Hc colocalizes extensively with LC3 along the axon tracks and in the soma. A–D, Hippocampal neurons cultured in microfluidic devices were incubated with BoNT/A-Hc-Atto647 (100 nm, Hc/A) for 5 min in high K+, followed by a 2–4 h chase in culture medium. Cells were then fixed and processed for immunofluorescence labeling of either LAMP1 or LC3 in the soma (A) and terminal (B) chambers. Scale bar, 10 μm. C, Neurons were treated as indicated before a 2–4 h chase, fixation, and labeling of the axon channels. Arrowheads indicate colocalized compartments. Scale bar, 5 μm. D, Relative distribution of LC3 and LAMP1-label compartments in the soma and nerve terminals. E, Level of colocalization between BoNT/A-Hc-Atto647 (Hc/A) and either LC3 or LAMP1 in soma and terminal chambers. Note the high level of colocalization of BoNT/A-Hc with LAMP1 in the soma, whereas in the terminal chamber, BoNT/A-Hc is mainly associated with LC3 compartments. Data are from 3 independent experiments; 15–49 fields are analyzed for each group. **p < 0.01; ***p < 0.001, Student's t test. F, G, Level of colocalization (Mander's analysis) between BoNT/A-Hc-Atto647 and LC3 in either low K+-or high K+-treated neurons quantified in the axon channels in the indicated conditions; n = 30 for low K+, n = 25 for high K+. ***p < 0.001, Mann–Whitney test.
- Figure 8.
Retrogradely transported mCherry-BoNT/A-Hc accumulates in lysosomes from spinal cord motor neuron soma after paw injection. A. Schematic of paw injection of mCherry-BoNT/A-Hc (Hc/A) in vivo. Twenty-four hours after injection, mice were killed and perfused. The spinal cord was then dissected and sliced for immunofluorescence processing. The level of colocalization of mCherry-BoNT/A-Hc wi‘th LC3 or LAMP1 was assessed in the cell soma of ipsilateral spinal cord motor neurons. B, C, Representative images of mCherry-BoNT/A-Hc colocalized with LC3 (B) and LAMP1 (C) in the soma of spinal cord motor neurons. Scale bar, 10 μm. D, E, Mander's ratio of colocalization between BoNT/A-Hc and LAMP1 or LC3, respectively. Data are from 3 independent experiments; n = 52 for LC3 staining, n = 59 for LAMP1 staining. ***p < 0.001, Student's t test.
- Figure 9.
Presynaptic activity increases LC3-mediated BoNT/A-Hc axonal retrograde trafficking. GFP-LC3- or RFP-LC3-transfected neurons cultured in microfluidic devices were incubated with BoNT/A-Hc-Atto647 (100 nm) for 5 min in either low- or high K+ medium, followed by a 2–4 h chase in culture. A, Representative images of axons extending into the nerve terminal chambers labeled with both BoNT/A-Hc-Atto647 (Hc/A, middle) and GFP-LC3 (top); arrowhead points to a soma. Time-lapse imaging was performed in regions proximal to the soma chamber (yellow boxes). Scale bar, 30 μm. B, Top, Time-lapse imaging of BoNT/A-Hc-Atto647 (Hc/A) and GFP-LC3 in axons. Bottom, Kymographs of the same axons showing carrier movement over 276 s. Arrowheads point to moving carriers. C, Top, Time-lapse imaging of BoNT/A-Hc-Atto647 (Hc/A) and RFP-LC3 in axons. Bottom, Kymographs of the same axons showing carrier movement over 264 s. Arrowheads point to moving carriers. Scale bars: x-axis, 10 μm; y-axis, 10 s. D–G, Quantification of the number of XFP-LC3 (GFP-LC3 or RFP-LC3, respectively, as labeled on the x-axis; D), BoNT/A-Hc-Atto647 (E), and BoNT/A-Hc/XFP-LC3 double-positive vesicles (F) detected over 400 s in the indicated conditions. G, Percentage of XFP-LC3 vesicles containing BoNT/A-Hc. GFP-LC3-transfected groups: n = 43 for low K+, n = 51 for high K+. RFP-LC3-transfected groups: n = 27 for low K+, n = 30 for high K+. *p < 0.05; **p < 0.01; ***p < 0.001, Mann–Whitney test.
- Figure 10.
Presynaptic activity does not influence the fate of retrogradely transported BoNT/A-Hc upon reaching the soma. Tandem fluorescently tagged LC3 (tFP-LC3: mRFP-EGFP-LC3)-transfected neurons were incubated with BoNT/A-Hc-Atto647 (100 nm) for 5 min in either low or high K+ medium, followed by a 2–4 h chase in culture medium. A, Representative images of autophagosome (Autophag. A1 and A3 boxes) and autolysosome (Autolyso., A2 and A4 boxes) and in the proximal axons (A1, A3) and soma (A2, A4) in the indicated conditions. Arrowheads indicate tFP-LC3-positive vesicles. Scale bar, 10 μm. B, Quantification of the number of somatic autolysosomes and autophagosomes in the indicated conditions. C, Percentage of somatic autophagosomes and autolysosomes containing BoNT/A-Hc in the indicated conditions. Data are from 3 independent cultures; n = 9 for low K+; n = 11 for high K+. *p < 0.05, Mann–Whitney test.
- Figure 11.
Activity-dependent BoNT/A-Hc retrograde flux is blocked by autophagy inhibitors. Hippocampal neurons cultured in microfluidic devices were incubated with wortmannin (40 nm), bafilomycin A1 (50 nm), or DMSO for 0.5 h before pulse-chase BoNT/A-Hc labeling at nerve terminals under high K+ stimulation. A, Kymographs of BoNT/A-Hc carriers in DMSO vehicle- (left), bafilomycin A1- (middle), or wortmannin (right)-treated axon channels over 174 s. Retrograde tracks are marked with arrowheads. Scale bars: x-axis, 10 μm; y-axis, 20 s. B, Quantification of BoNT/A-Hc retrograde carrier numbers in corresponding neurons, as shown in A; n = 53 DMSO; n = 20 bafilomycin A1; n = 56 wortmannin, ***p < 0.001, Mann–Whitney test.
