Intramuscular Botulinum Neurotoxin Serotypes E and A Elicit Distinct Effects on SNAP25 Protein Fragments, Muscular Histology, Spread and Neuronal Transport: An Integrated Histology-Based Study in the Rat

Abstract

Botulinum neurotoxins E (BoNT/E) and A (BoNT/A) act by cleaving Synaptosome-Associated Protein 25 (SNAP25) at two different C-terminal sites, but they display very distinct durations of action, BoNT/E being short acting and BoNT/A long acting. We investigated the duration of action, spread and neuronal transport of BoNT/E (6.5 ng/kg) and BoNT/A (125 pg/kg) after single intramuscular administrations of high equivalent efficacious doses, in rats, over a 30- or 75-day periods, respectively. To achieve this, we used (i) digit abduction score assay, (ii) immunohistochemistry for SNAP25 (N-ter part; SNAP25^N-ter and C-ter part; SNAP25^C-ter) and its cleavage sites (cleaved SNAP25; c-SNAP25^E and c-SNAP25^A) and (iii) muscular changes in histopathology evaluation. Combined in vivo observation and immunohistochemistry analysis revealed that, compared to BoNT/A, BoNT/E induces minimal muscular changes, possesses a lower duration of action, a reduced ability to spread and a decreased capacity to be transported to the lumbar spinal cord. Interestingly, SNAP25^C-ter completely disappeared for both toxins during the peak of efficacy, suggesting that the persistence of toxin effects is driven by the persistence of proteases in tissues. These data unveil some new molecular mechanisms of action of the short-acting BoNT/E and long-acting BoNT/A, and reinforce their overall safety profiles.

Keywords: SNAP25; botulinum neurotoxin type A; botulinum neurotoxin type E; histopathology; immunohistochemistry; muscle; rat; spinal cord.

Figure 1

Intramuscular injection of BoNT/E and BoNT/A in rat gastrocnemius impacted differently DAS assay. (A) Quantification of DAS in left hind-paw after intramuscular injection of 6.5 ng/kg BoNT/E or GPB (controls) in left gastrocnemius. (B) Quantification of DAS in left hind-paw after intramuscular injection of 125 pg/kg BoNT/A or gelatin phosphate buffer (GPB, controls) in left gastrocnemius. Data are represented as mean ± SEM of n = 3 to 24 rats for BoNT/E and BoNT/A, from D1 to D30 and D1 to D75, respectively.

Figure 2

Representation of rat SNAP25 protein, cleavage sites of BoNT/E and BoNT/A and regions targeted by antibodies used in this work, and representative images of c-SNAP25^E and SNAP25^C-ter staining in rat muscle. (A) BoNT/E and BoNT/A cleave SNAP25 at C-ter part, at R180-I181 and Q197-R198, respectively. Antibody 111 011 recognizes both noncleaved and cleaved forms of SNAP25 N-ter part (SNAP25^N-ter), EF14007 and EF13002 specifically recognize BoNT/E- and BoNT/A-cleaved SNAP25 (c-SNAP25^E, c-SNAP25^A), respectively, and 6C11 recognizes SNAP25 C-ter part located right after BoNT/A and BoNT/E cleavage sites (SNAP25^C-ter). In vehicle-treated rat muscle, c-SNAP25^E was absent in nerve bundle (B) and at neuromuscular junction (C), but was observed in BoNT/E-treated rat muscle (D,E). SNAP25^C-ter was present in nerve bundle (F) and at neuromuscular junction (G) in vehicle-treated rat muscle, but progressively disappeared in BoNT/E- and BoNT/A-treated rat muscle (H,I). Scale bars; 20 µm, except for (D,E); 50 µm.

Figure 3

Kinetics and amplitude of c-SNAP25^E and c-SNAP25^A staining in injected, adjacent and contralateral muscles were different after BoNT/E or BoNT/A administration in rat gastrocnemius. (A) Representation of a vehicle-treated rat hindleg cross-sectioned at the level of the gastrocnemius. Injected (gastrocnemius) and adjacent (ipsilateral peroneus, EDL and tibialis) muscles are identified. Scale bar; 4 mm. (B) Quantification of c-SNAP25^E staining in injected, adjacent and contralateral muscles, after intramuscular injection of 6.5 ng/kg BoNT/E in left gastrocnemius. (C) Quantification of c-SNAP25^A staining in injected, adjacent and contralateral muscles, after intramuscular injection of 125 pg/kg BoNT/A in left gastrocnemius. Data are represented as mean ± SEM of n = 3 rats per time point. Vehicle-treated animals did not show any c-SNAP25^E or c-SNAP25^A staining throughout this study and were not represented.

Figure 4

Evolution of SNAP25^C-ter staining was inversely proportional to the one of c-SNAP25^E and c-SNAP25^A in injected and adjacent muscles after BoNT/E or BoNT/A administration in rat gastrocnemius. Quantification of c-SNAP25^E, SNAP25^N-ter and SNAP25^C-ter in injected gastrocnemius (A), ipsilateral EDL (C) and ipsilateral tibialis (E) after intramuscular injection of 6.5 ng/kg BoNT/E in left gastrocnemius. Quantification of c-SNAP25^A, SNAP25^N-ter and SNAP25^C-ter in injected gastrocnemius (B), ipsilateral EDL (D) and ipsilateral tibialis (F) after intramuscular injection of 125 pg/kg BoNT/A in left gastrocnemius. Data are represented as mean ± SEM of n = 3 (BoNT/E or BoNT/A) rats per time point or n = 5 (vehicle). Cont.; control (vehicle-treated) animals.

Figure 5

Intramuscular injection of BoNT/E or BoNT/A in rat gastrocnemius impacted myofiber sizes differently. (A) Quantification of myofiber size using image analysis (histograms and table) or histopathology (line graphs) in injected gastrocnemius after intramuscular injection of 6.5 ng/kg BoNT/E. (B) Quantification of myofiber size using image analysis (histograms and table) or histopathology (line graphs) in injected gastrocnemius after intramuscular injection of 125 pg/kg BoNT/A. Data are represented as the mean of the proportion of myofibers in each size category of n = 3 (BoNT/E or BoNT/A) rats per time point or n = 5 (vehicle) (histograms), or as mean ± SEM of n = 3 (BoNT/E or BoNT/A) rats per time point (line graphs). Control; vehicle-treated animals. (C) Representative images of reticulin staining allowing the segmentation of myofibers in injected gastrocnemius after intramuscular injection of 6.5 ng/kg BoNT/E. (D) Representative images of reticulin staining allowing the segmentation of myofibers in injected gastrocnemius after intramuscular injection of 125 pg/kg BoNT/A. Scale bars; 50 µm for Magnification ×400 and 20 µm for Magnification ×1000.

Figure 6

Representative images of histopathological lesions observed using Masson’s Trichrome staining in injected gastrocnemius after intramuscular injection of 125 pg/kg BoNT/A. (A) Normal aspect of myofibers in vehicle-treated rat. (B) Moderate myofiber atrophy (BoNT/A, D20). (C) Increased myofiber size with rounded aspect (BoNT/A, D75). (D) Myofiber cytoplasmic pallor, most likely due to myofilament autophagy. Insert: central nuclei in atrophied myofibers (BoNT/A, D20). (E) Reappearance of cytoplasmic striations (black arrowhead, BoNT/A, D27). (F,G) Myofiber degeneration with cytoplasmic fragmentation (BoNT/A, D20). (H) Myofiber atrophy with fatty changes; cytoplasmic-clear vacuoles, most likely lipidic (BoNT/A, D75). Scale bars; 20 µm.

Figure 7

Intramuscular injection of BoNT/E or BoNT/A in rat gastrocnemius triggered distinct SNAP25 cleavage in the lumbar spinal cord without histopathological changes. Representative images of H&E staining on lumbar spinal cord sections from rats injected intramuscularly in the left gastrocnemius with vehicle (A–C), 6.5 ng/kg BoNT/E (D–F) or 125 pg/kg BoNT/A (K–M). Representative images of SNAP25 cleavage and its quantification in lumbar spinal cord sections from rats injected intramuscularly in the left gastrocnemius with 6.5 ng/kg BoNT/E (G–J) or 125 pg/kg BoNT/A (N–Q). Data are represented as mean ± SEM of n = 3 rats per time point. Vehicle-treated animals did not show any c-SNAP25^E or c-SNAP25^A staining throughout the study and were not represented. Scale bars; 500 µm for Magnification ×40, 100 µm for Magnification ×200 and 20 µm for Magnification ×1000.