The mechanism of action of botulinum neurotoxin type A (BoNT/A) is well characterized, but some published evidence suggests the potential for neuronal retrograde transport and cell-to-cell transfer (transcytosis) under certain experimental conditions. The present study evaluated the potential for these processes using a highly selective antibody for the BoNT/A-cleaved substrate (SNAP25197) combined with 3-dimensional imaging. SNAP25197 was characterized in a rat motor neuron (MN) pathway following toxin intramuscular injections at various doses to determine whether SNAP25197 is confined to MNs or also found in neighboring cells or nerve fibers within spinal cord (SC). Results demonstrated that SNAP25197 immuno-reactive staining was colocalized with biomarkers for MNs, but not with markers for neighboring neurons, nerve fibers or glial cells. Additionally, a high dose of BoNT/A, but not a lower dose, resulted in sporadic SNAP25197 signal in distal muscles and associated SC regions without evidence for transcytosis, suggesting that the staining was due to systemic spread of the toxin. Despite this spread, functional effects were not detected in the distal muscles. Therefore, under the present experimental conditions, our results suggest that BoNT/A is confined to MNs and any evidence of distal activity is due to limited systemic spread of the toxin at higher doses and not through transcytosis within SC. Lastly, at higher doses of BoNT/A, SNAP25197 was expressed throughout MNs and colocalized with synaptic markers on the plasma membrane at 6 days post-treatment. These data support previous studies suggesting that SNAP25197 may be incorporated into SNARE-protein complexes within the affected MNs.

Background: Expression quantitative trait locus (eQTL) mapping is used to find loci that are responsible for the transcriptional activity of a particular gene. In recent eQTL studies, expression profiles were derived from either homogenized whole brain or collections of large brain regions. However, the brain is a very heterogeneous organ, and expression profiles of different brain regions vary significantly. Because of the importance and potential power of eQTL studies in identifying regulatory networks, we analyzed gene expression patterns in different brain regions from multiple inbred mouse strains and investigated the implications for the design and analysis of eQTL studies. Results: Gene expression profiles of five brain regions in six inbred mouse strains were studied. Few genes exhibited a significant strain-specific expression pattern, whereas a large number of genes exhibited brain region-specific patterns. We constructed phylogenetic trees based on the expression relationships between the strains and compared them with a DNA-level relationship tree. The trees based on the expression of strain-specific genes were constant across brain regions and mirrored DNA-level variation. However, the trees based on region-specific genes exhibited a different set of strain relationships, depending on the brain region. An eQTL analysis showed enrichment of cis-acting regulators among strain-specific genes, whereas brain region-specific genes appear to be mainly regulated by trans-acting elements. Conclusion: Our results suggest that many regulatory networks are highly brain region specific and indicate the importance of conducting eQTL mapping studies using data from brain regions or tissues that are physiologically and phenotypically relevant to the trait of interest.

Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A’s location, distribution, and movement within a cell. Due to BoNT/A’s high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25₁₉₇) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25₁₉₇, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25₁₉₇ rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25₁₉₇ in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25₁₉₇-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.

Differences in botulinum neurotoxin manufacturing, formulation, and potency evaluation can impact dose and biological activity, which ultimately affect duration of action. The potency of different labeled vials of incobotulinumtoxinA (Xeomin^®; 50 U, 100 U, or 200 U vials; incobotA) versus onabotulinumtoxinA (BOTOX^®; 100 U vial; onabotA) were compared on a unit-to-unit basis to assess biological activity using in vitro (light-chain activity high-performance liquid chromatography (LCA-HPLC) and cell-based potency assay (CBPA)) and in vivo (rat compound muscle action potential (CMAP) and mouse digit abduction score (DAS)) assays. Using LCA-HPLC, incobotA units displayed approximately 54% of the protease activity of label-stated equivalent onabotA units. Lower potency, reflected by higher EC₅₀, ID₅₀, and ED₅₀ values (pooled mean ± SEM), was displayed by incobotA compared to onabotA in the CBPA (EC₅₀: incobotA 7.6 ± 0.7 U/mL; onabotA 5.9 ± 0.5 U/mL), CMAP (ID₅₀: incobotA 0.078 ± 0.005 U/rat; onabotA 0.053 ± 0.004 U/rat), and DAS (ED₅₀: incobotA 14.2 ± 0.5 U/kg; onabotA 8.7 ± 0.3 U/kg) assays. Lastly, in the DAS assay, onabotA had a longer duration of action compared to incobotA when dosed at label-stated equivalent units. In summary, onabotA consistently displayed greater biological activity than incobotA in two in vitro and two in vivo assays. Differences in the assay results do not support dose interchangeability between the two products.

OnabotulinumtoxinA (BoNT-A) is an Food and Drug Administration-approved, peripherally acting preventive migraine drug capable of inhibiting meningeal nociceptors. Expanding our view of how else this neurotoxin attenuates the activation of the meningeal nociceptors, we reasoned that if the stimulus that triggers the activation of the nociceptor is lessened, the magnitude and/or duration of the nociceptors' activation could diminish as well. In the current study, we further examine this possibility using electrocorticogram recording techniques, immunohistochemistry, and 2-photon microscopy. We report (1) that scalp (head) but not lumbar (back) injections of BoNT-A shorten the period of profound depression of spontaneous cortical activity that follows a pinprick-induced cortical spreading depression (CSD); (2) that neither scalp nor lumbar injections prevent the induction, occurrence, propagation, or spreading velocity of a single wave of CSD; (3) that cleaved SNAP25—one of the most convincing tools to determine the anatomical targeting of BoNT-A treatment—could easily be detected in pericranial muscles at the injection sites and in nerve fibers of the intracranial dura, but not within any cortical area affected by the CSD; (4) that the absence of cleaved SNAP25 within the cortex and pia is unrelated to whether the blood–brain barrier is intact or compromised; and (5) that BoNT-A does not alter vascular responses to CSD. To the best of our knowledge, this is the first report of peripherally applied BoNT-A's ability to alter a neuronal function along a central nervous system pathway involved in the pathophysiology of migraine.