Fig 1.
Characterizing triple point-mutations and generation of Syt1M3 and Syt2M3 KI mice.
A. Sequence alignment of the BoNT-binding region in Syt1 and Syt2 (residues 32 to 52 in Syt1 and 40 to 60 in Syt2). Residues highlighted in red mark the designed triple mutations (Syt1M3 and Syt2M3) that abolish BoNT-binding. B. Co-crystal structure of the BoNT/B-Syt2 complex (PDB:4KBB) with the triple mutation sites (F54A, F55A, E57K) highlighted. C-E. Miniature inhibitory postsynaptic currents (mIPSC) were monitored and analyzed by whole-cell patch-clamp recording in cultured rat cortical neurons, with representative traces shown in C, frequency (freq) in D, and amplitude (amp) in E. WT: wild type neurons; Syt1 KD: neurons with their endogenous Syt1 knocked down by shRNA expressed via lentiviral transduction; Syt1WT: WT Syt1 is expressed in Syt1 KD neurons via lentiviral transduction; Syt1M3: Syt1M3 mutant was expressed in Syt1 KD neurons via lentiviral transduction. Syt1 KD increased mIPSC frequency, which is restored by expression of either Syt1WT or Syt1M3. For each condition, we recorded from 12–15 neurons from a total of four coverslips. Data shown are means ± SEM. Statistical analysis was performed with Student’s t-test (***P < 0.001). F-H. Spontaneous inhibitory postsynaptic currents (sIPSC) were monitored and analyzed by whole-cell patch-clamp recording in cultured rat cortical neurons, with representative traces shown in F, frequency in G, and amplitude in H. The amplitude and frequency of sIPSC were greatly deceased in Syt1 KD, and both Syt1WT and Syt1M3 restored normal levels of sIPSC. Data shown are means ± SEM. Statistical analysis was performed with Student’s t-test (***P < 0.001). I-K. Evoked inhibitory postsynaptic currents (IPSC) were monitored and analyzed by whole-cell patch-clamp recording in cultured rat cortical neurons, with representative traces shown in I, amplitude in J, and total charge transfer in K. The amplitude and charge of IPSC were greatly deceased in Syt1 KD, and both Syt1WT and Syt1M3 restored normal levels of IPSC. L. Schematic drawing of generating Syt1 and Syt2 triple mutation KI mice via CRISPR-Cas9 approach. M. Expression levels of synaptic vesicle proteins Syt1, Syt2, and SV2, and toxin substrate proteins SNAP-25, VAMP-2, Syntaxin-1, analyzed by immunoblot of brain lysates, remain unchanged in Syt1M3 KI and Syt2M3 KI mice compared with WT mice.
Fig 2.
Syt2 and Syt1 are differentially expressed at the mouse diaphragm and bladder.
A. Immunohistochemistry analysis detected expression of Syt2, but not Syt1, in motor nerve terminals at WT mouse diaphragm neuromuscular junctions (NMJs). SMI-312 antibody detects neurofilament and marks the phrenic nerve. α-Bungarotoxin (a-btx) labels post-synaptic acetylcholine receptors and serves as a marker for NMJs. Scale bar, 20 μm. B. Immunohistochemistry analysis of bladder sections from WT mice detected expression of Syt1, but not Syt2 in neurons. β-3 tubulin marks nerve fibers and synapsin marks the pre-synaptic site of neuronal varicosities in bladder tissues. Scale bar, 20 μm. C. Diaphragms isolated from WT, Syt1M3 KI, or Syt2M3 KI mice were incubated with BoNT/B (100 nM, 90 min) in high K+ buffer. Tissues were washed, and immunohistochemistry analysis was performed on whole mount tissues. BoNT/B binding and entry was detected on both WT and Syt1M3, but not Syt2M3 diaphragms. D. Bladder tissues isolated from WT, Syt1M3, or Syt2M3 mice were incubated with BoNT/B (100 nM, 90 min) in high K+ buffer. Tissues were washed, and immunohistochemistry analysis was performed. BoNT/B binding and entry was detected on both WT and Syt2M3, but not Syt1M3 bladders. Representative images were selected from n = 3–4 biological replicates.
Fig 3.
BoNT/B showed reduced potency and toxicity on Syt2M3 KI mice.
A-C. BoNT/A (5 pg) was injected into the right gastrocnemius muscle of mice and muscle paralysis was scored by monitoring the degree of toe spreading in startle responses (DAS assays). BoNT/A showed the same levels of potency in inducing local muscle paralysis in DAS assays in WT, Syt1M3, Syt2M3, and Syt1/Syt2M3 double KI mice. Representative pictures of toe spreading are shown in A. The maximal DAS scores are plotted in B, and the mean daily DAS scores three days after toxin injection are plotted in C. Data represent mean +/- SEM. ns = not significant. D-F. BoNT/B showed reduced potency in inducing local paralysis and systemic toxicity in DAS assays on Syt2M3 KI and Syt1/Syt2M3 KI mice compared with WT and Syt1M3 KI mice. Representative images of toe spreading in DAS assays with the indicated doses are shown in D, mean DAS scores with different toxin doses are plotted in E, and the survival rates with the indicated toxin doses are plotted in F. The BoNT/B doses that resulted in moribund start at 4.25 pg for WT, 3.25 pg for Syt1M3, 125 pg for Syt2M3, and 200 pg for Syt1/Syt2M3 mice. Panels A, B, C–WT and Syt1M3 mice, n = 3, Syt2M3 and Syt1/Syt2M3 mice, n = 4. Panels D, E–all groups that received BoNT/B injection, n = 5. Except for the following groups: WT 5.5 pg BoNT/B (n = 4); Syt2M3 100 pg BoNT/B (n = 10); Syt2M3 150 pg BoNT/B (n = 3); Syt1/Syt2M3 100 pg BoNT/B (n = 6); Syt1/Syt2M3 150 pg BoNT/B (n = 6). Panel F–these numbers are from the same dataset as panels D, E.
Table 1.
A list of toxin doses correlating with systemic toxicity in WT and KI mice.
The full ranges of titration toxin doses and survival rates are shown in Figs 3F, 4B and 4D.
Fig 4.
BoNT/G and BoNT/DC showed reduced potency and toxicity on Syt2M3 KI mice.
A-B. BoNT/G showed reduced potency in inducing local muscle paralysis in DAS assays in Syt2M3 and Syt1/Syt2M3 mice (A), as well as reduced toxicity in systemic lethality levels (B), compared with WT and Syt1M3 mice. The BoNT/G doses that induced moribund are 2.3 ng in both WT and Syt1M3 mice, and 75 ng in Syt2M3 and Syt1/Syt2M3 mice. C-D. BoNT/DC did not elicit significant local paralysis before it caused death of mice in DAS assays. It showed reduced toxicity in Syt2M3 and Syt1/Syt2M3 mice compared with WT and Syt1M3 mice. The BoNT/DC doses that induced moribund are at 25.6 pg in WT mice, 20.5 pg in Syt1M3 mice, and 275 pg in both Syt2M3 and Syt1/Syt2M3 mice. Panels A and B, n = 5; panels C and D, n = 5, except for the following: Syt2M3, BoNT/DC 200 pg (n = 4); Syt2M3, BoNT/DC 275 pg (n = 4); Syt2M3, BoNT/DC 325 pg (n = 3); Syt2M3, BoNT/DC 425 pg (n = 3); Syt1/Syt2M3, BoNT/DC 200 pg (n = 4); Syt1/Syt2M3, BoNT/DC 250 pg (n = 4); Syt1/Syt2M3, BoNT/DC 275 pg (n = 4); Syt1/Syt2M3, BoNT/DC 325 pg (n = 4); Syt1/Syt2M3, BoNT/DC 400 pg (n = 4); Syt1/Syt2M3, BoNT/DC 500 pg (n = 3).
Fig 5.
Syt1M3 bladder strips are less sensitive to BoNT/B in ex vivo bladder contraction assays.
A. Schematic diagram of ex vivo bladder strip contraction assay. Bladder strips were immersed in aerated Krebs solution. Exogenous pharmacologic agents (i.e., carbachol, BoNT/B) were added directly to the solution. Trains of voltage delivered over a range of frequencies were generated by the electrical field stimulator to trigger bladder contraction, which is detected and measured by the force transducer. B. Bladder weights across WT and KI mice were similar prior to ex vivo contraction assay. ns = not significant (ANOVA). C-D. The nerve-evoked contraction forces of WT (n = 3), Syt1M3 (n = 5), and Syt2M3 (n = 4) bladder strips were recorded, with the representative force trace shown in C. A set of frequency-dependent stimulations (enclosed by brackets above tracing) were delivered every 100 min. After a baseline frequency-response, evoked responses at constant frequency (16 Hz) were produced every 15 min. BoNT/B (0.3 nM) was administered at red arrow. The time dependent effects of BoNT/B on nerve evoked responses are shown in frequency-response curves that were plotted in 100 min intervals, shown in D. Incubation with BoNT/B reduced contractions of WT and Syt2M3 bladders, but did not affect Syt1M3 bladders. *p<0.05, repeated measures ANOVA. E. Carbachol induces direct muscle contraction independent of neurotransmission. Carbachol does-response curves were generated at the beginning and end of the experiment as a control showing that bladder strips maintained their viability and contractility after incubation with BoNT/B for 300 min. p>0.05, RM ANOVA.
Fig 6.
Analyzing BoNT-induced urinary retention in vivo.
A. Schematic illustration of the bladder injection model. Mice were anesthetized with isoflurane and a low midline laparotomy was made. The bladder was exteriorized and decompressed of urine to thicken the bladder wall. BoNT/B was injected into the lateral walls of the bladder bilaterally. B. Mice injected with BoNT/B were subjected to 4-hour nighttime voiding spot assay every 24 hours for 4 days. Representative images for urine spots (black areas) are shown. C. Voiding spots quantified into total void areas (inch2) demonstrate similar pre-injection urine volumes across WT, Syt1M3, and Syt2M3 mice (n = 5 for each group). All groups showed a decline in voided volumes in sham controls that went through the surgery and injection procedures without toxins, likely from surgical manipulation of the bladder and the use of post-operative pain control narcotics. n = 3 for each group. D. At 24–48 hours post-injection, WT and Syt2 KI mice had significantly diminished voided volumes compared to Syt1 KI mice, with progressive recovery over 96 hours. *P<0.05, **P<0.005, ns = not significant. E. Number of voiding spots per 4-hour nighttime evaluation showed no difference across genotypes, and across pre- and post-injection time points, demonstrating that voiding frequency is unchanged across groups. ns = not significant.