- Email: [email protected]
Botulinum toxin in the treatment of upper limb spasticity
S8
Abstracts / Toxicon 123 (2016) S2eS90
mechanism (increased presynaptic release probabilities at non-intoxicated release sites); and a BoNT/A-specific mechanism. We will describe the clinical and molecular implications of these findings in terms of therapeutically accessible stages of disease, as well as posit novel therapeutic approaches. Keywords: 3,4-Diaminopyridine; Diaphragm paralysis; Endplate potentials; Toxin-resistant neurotransmission
18. BOTULINUM NEUROTOXIN TYPE AeCLEAVED SNAP25 IS CONFINED TO PRIMARY MOTOR NEURONS AND EXPRESSED ON THE PLASMA MEMBRANE FOLLOWING INTRAMUSCULAR TOXIN INJECTION Ron S. Broide a, *, Brian B. Cai a, Joseph Francis a, Mitchell F. Brin a, b. a Department of Biological Sciences, Allergan, Irvine, CA, USA; b Department of Neurology, University of California, Irvine, CA, USA * Corresponding author: Department of Biological Sciences, Allergan, 2525 Dupont Drive, Irvine, CA, USA. E-mail address:[email protected].
Fig. 1. (A) The effect of DAP treatment on contraction of BoNT/A-treated diaphragms is inverse to the degree of paralysis. (Inset) Exemplar traces of individual twitches immediately prior to DAP addition (left) and 20 min after DAP addition (right) at various stages of disease. All amplitudes are normalized to a baseline twitch recorded immediately prior to any treatment. (B) DAP treatment restores persistent, low-amplitude endplate potentials (EPPs) in continuous recordings from BoNT/A-intoxicated endplates (n ¼ 3 endplates).
17. BOTULINUM TOXIN IN THE TREATMENT OF UPPER LIMB SPASTICITY Allison Brashear. Department of Neurology, Wake Forest School of Medicine, 475 Vine Street, Winston-Salem, NC 27101, USA. E-mail address:[email protected] Botulinum toxin in the treatment of upper extremity spasticity is generally accepted as first-line therapy. In the United States (US), there are three forms of botulinum toxin type A approved for treatment of spasticity. Ongoing trials focus on outcome measures, long-term use and dosing. A recent publication by the American Academy of Neurology in the journal Neurology entitled “Practice guideline update summary: Botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity, and headache: Report of the Guideline Development Subcommittee of the American Academy of Neurology” (Simpson 2016), concluded that all three forms of botulinum toxin type A are established as effective and should be offered. It also noted that the serotype B product is probably effective and should be considered for upper limb spasticity. In addition, each of the type A serotypes have reported large multicenter trials demonstrating efficacy in improving tone compared to placebo. Each of these large trials also used a measure of clinical meaningfulness that included the global impression of change scale and/or the Disability Assessment Scale (DAS). The results of these large trials demonstrate that the reduction of tone is clinically meaningful for patients. However, these products are dosed differently and cannot be interchanged. Meaningful comparison of products has not been done. Attention has not been paid to dosing and technique of localization in many of these trials, though the results demonstrate that several products are effective in spasticity compared to placebo. Clinicians will need to educate themselves on the results of the trials for each product and understand the different dosing regimens. It is unlikely that large detailed studies on localization and dosing will be performed to answer some of the more complex questions that those who treat patients on a recurring basis are asking. In the US, the focus on cost of care, outcome measures, and patient expectations may begin to drive more of the research questions. It will be incumbent on the research community to demonstrate the impact of the injections on the care of the patient and the caregiver, as well as the overall impact on function relative to the cost of the procedures. Reference Simpson DM, Hallett M, Ashman EJ, et al. Practice guideline update summary: botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity, and headache: Report of the Guideline Development Subcommittee of the American Academy of Neurology. Neurology. 2016;86(19):818-826.
Introduction and objectives: Botulinum neurotoxin type A (BoNT/A) is used clinically for a growing number of indications. The present study was performed to assess the potential for neuronal transport and transcytosis of BoNT/A (onabotulinumtoxinA) following peripheral administration to the rat tibialis anterior hind limb muscle. The expression of BoNT/A-cleaved substrate (SNAP25197) was characterized in vivo in a rat motor neuron (MN) pathway following toxin intramuscular injections at relatively low and high doses. Methods: Using a highly selective anti-SNAP25197 antibody combined with quantitative, high-resolution 3D imaging, we performed a systematic evaluation to determine whether SNAP25197 is confined to primary MNs or is also found in neighboring cells or nerve fibers within the ventrolateral spinal cord (SC). Results: The results demonstrated that SNAP25197-immunoreactive (IR) staining is co-localized with biomarkers for MNs but not co-localized with markers for neighboring neurons, nerve fibers, or glial cells. Additionally, we found that an experimentally defined high dose of BoNT/A (30 U/kg), but not a lower dose (3 U/kg), resulted in distal spread of toxin activity, giving rise to sporadic SNAP25197 signal in distal muscles and associated SC regions without evidence of transcytotic (cell-to-cell) migration. Despite the spread in activity, functional effects, as measured by the Digit Abduction Score assay, were not detected in the distal muscles. Last, we discovered that at higher BoNT/A doses, SNAP25197-IR staining was expressed throughout the MN and was highly co-localized with synaptic markers on the plasma membrane at 6 days posttreatment. Conclusions: The initial results indicate that BoNT/A is confined to primary MNs and that any evidence of distal activity is a result of limited systemic spread of the toxin at higher doses and not through transcytosis within the SC. The final data support previous studies suggesting that SNAP25197 acts as a dominant-negative soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein within the affected MNs. Keywords: Cholinergic; Motor neuron; Neuromuscular junction; OnabotulinumtoxinA; SNAP25; Spinal cord 19. ASSEMBLY AND DISASSEMBLY OF THE SNARE COMPLEX Axel T. Brunger. Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA. E-mail address:[email protected] We determined the structure of the so-called 20S complex consisting of the neuronal soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex, alpha soluble NSF attachment protein (a-SNAP), and the adenosine triphosphatase (ATPase) NSF by electron cryomicroscopy at a resolution of 7.6 Å. This structure along with the structures of NSF alone in different nucleotide states have provided the first glimpses of how this SNARE-recycling machine works. The SNARE complex resembles a rope with a left-handed twist, and NSF uses SNAPs to grasp the “rope” in multiple places. The SNAPs wrap around the SNARE complex with a right-handed twist, suggesting that the disassembly occurs via a simple unwinding motion that frees the zipped SNARE proteins. We also determined the first atomic-resolution structure of the complex between the Ca2+ sensor synaptotagmin-1 and the neuronal SNARE complex. This structure revealed a conserved, Ca2+-independent interface that is essential for fast synchronous neurotransmitter release, suggesting that the synaptotagmin/SNARE complex is preassembled prior to the arrival of Ca2+, and moves en bloc as an entity upon triggering by Ca2+. The simultaneous membrane interactions of the Ca2+-
Abstracts / Toxicon 123 (2016) S2eS90
mechanism (increased presynaptic release probabilities at non-intoxicated release sites); and a BoNT/A-specific mechanism. We will describe the clinical and molecular implications of these findings in terms of therapeutically accessible stages of disease, as well as posit novel therapeutic approaches. Keywords: 3,4-Diaminopyridine; Diaphragm paralysis; Endplate potentials; Toxin-resistant neurotransmission
18. BOTULINUM NEUROTOXIN TYPE AeCLEAVED SNAP25 IS CONFINED TO PRIMARY MOTOR NEURONS AND EXPRESSED ON THE PLASMA MEMBRANE FOLLOWING INTRAMUSCULAR TOXIN INJECTION Ron S. Broide a, *, Brian B. Cai a, Joseph Francis a, Mitchell F. Brin a, b. a Department of Biological Sciences, Allergan, Irvine, CA, USA; b Department of Neurology, University of California, Irvine, CA, USA * Corresponding author: Department of Biological Sciences, Allergan, 2525 Dupont Drive, Irvine, CA, USA. E-mail address:[email protected].
Fig. 1. (A) The effect of DAP treatment on contraction of BoNT/A-treated diaphragms is inverse to the degree of paralysis. (Inset) Exemplar traces of individual twitches immediately prior to DAP addition (left) and 20 min after DAP addition (right) at various stages of disease. All amplitudes are normalized to a baseline twitch recorded immediately prior to any treatment. (B) DAP treatment restores persistent, low-amplitude endplate potentials (EPPs) in continuous recordings from BoNT/A-intoxicated endplates (n ¼ 3 endplates).
17. BOTULINUM TOXIN IN THE TREATMENT OF UPPER LIMB SPASTICITY Allison Brashear. Department of Neurology, Wake Forest School of Medicine, 475 Vine Street, Winston-Salem, NC 27101, USA. E-mail address:[email protected] Botulinum toxin in the treatment of upper extremity spasticity is generally accepted as first-line therapy. In the United States (US), there are three forms of botulinum toxin type A approved for treatment of spasticity. Ongoing trials focus on outcome measures, long-term use and dosing. A recent publication by the American Academy of Neurology in the journal Neurology entitled “Practice guideline update summary: Botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity, and headache: Report of the Guideline Development Subcommittee of the American Academy of Neurology” (Simpson 2016), concluded that all three forms of botulinum toxin type A are established as effective and should be offered. It also noted that the serotype B product is probably effective and should be considered for upper limb spasticity. In addition, each of the type A serotypes have reported large multicenter trials demonstrating efficacy in improving tone compared to placebo. Each of these large trials also used a measure of clinical meaningfulness that included the global impression of change scale and/or the Disability Assessment Scale (DAS). The results of these large trials demonstrate that the reduction of tone is clinically meaningful for patients. However, these products are dosed differently and cannot be interchanged. Meaningful comparison of products has not been done. Attention has not been paid to dosing and technique of localization in many of these trials, though the results demonstrate that several products are effective in spasticity compared to placebo. Clinicians will need to educate themselves on the results of the trials for each product and understand the different dosing regimens. It is unlikely that large detailed studies on localization and dosing will be performed to answer some of the more complex questions that those who treat patients on a recurring basis are asking. In the US, the focus on cost of care, outcome measures, and patient expectations may begin to drive more of the research questions. It will be incumbent on the research community to demonstrate the impact of the injections on the care of the patient and the caregiver, as well as the overall impact on function relative to the cost of the procedures. Reference Simpson DM, Hallett M, Ashman EJ, et al. Practice guideline update summary: botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity, and headache: Report of the Guideline Development Subcommittee of the American Academy of Neurology. Neurology. 2016;86(19):818-826.
Introduction and objectives: Botulinum neurotoxin type A (BoNT/A) is used clinically for a growing number of indications. The present study was performed to assess the potential for neuronal transport and transcytosis of BoNT/A (onabotulinumtoxinA) following peripheral administration to the rat tibialis anterior hind limb muscle. The expression of BoNT/A-cleaved substrate (SNAP25197) was characterized in vivo in a rat motor neuron (MN) pathway following toxin intramuscular injections at relatively low and high doses. Methods: Using a highly selective anti-SNAP25197 antibody combined with quantitative, high-resolution 3D imaging, we performed a systematic evaluation to determine whether SNAP25197 is confined to primary MNs or is also found in neighboring cells or nerve fibers within the ventrolateral spinal cord (SC). Results: The results demonstrated that SNAP25197-immunoreactive (IR) staining is co-localized with biomarkers for MNs but not co-localized with markers for neighboring neurons, nerve fibers, or glial cells. Additionally, we found that an experimentally defined high dose of BoNT/A (30 U/kg), but not a lower dose (3 U/kg), resulted in distal spread of toxin activity, giving rise to sporadic SNAP25197 signal in distal muscles and associated SC regions without evidence of transcytotic (cell-to-cell) migration. Despite the spread in activity, functional effects, as measured by the Digit Abduction Score assay, were not detected in the distal muscles. Last, we discovered that at higher BoNT/A doses, SNAP25197-IR staining was expressed throughout the MN and was highly co-localized with synaptic markers on the plasma membrane at 6 days posttreatment. Conclusions: The initial results indicate that BoNT/A is confined to primary MNs and that any evidence of distal activity is a result of limited systemic spread of the toxin at higher doses and not through transcytosis within the SC. The final data support previous studies suggesting that SNAP25197 acts as a dominant-negative soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein within the affected MNs. Keywords: Cholinergic; Motor neuron; Neuromuscular junction; OnabotulinumtoxinA; SNAP25; Spinal cord 19. ASSEMBLY AND DISASSEMBLY OF THE SNARE COMPLEX Axel T. Brunger. Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA. E-mail address:[email protected] We determined the structure of the so-called 20S complex consisting of the neuronal soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex, alpha soluble NSF attachment protein (a-SNAP), and the adenosine triphosphatase (ATPase) NSF by electron cryomicroscopy at a resolution of 7.6 Å. This structure along with the structures of NSF alone in different nucleotide states have provided the first glimpses of how this SNARE-recycling machine works. The SNARE complex resembles a rope with a left-handed twist, and NSF uses SNAPs to grasp the “rope” in multiple places. The SNAPs wrap around the SNARE complex with a right-handed twist, suggesting that the disassembly occurs via a simple unwinding motion that frees the zipped SNARE proteins. We also determined the first atomic-resolution structure of the complex between the Ca2+ sensor synaptotagmin-1 and the neuronal SNARE complex. This structure revealed a conserved, Ca2+-independent interface that is essential for fast synchronous neurotransmitter release, suggesting that the synaptotagmin/SNARE complex is preassembled prior to the arrival of Ca2+, and moves en bloc as an entity upon triggering by Ca2+. The simultaneous membrane interactions of the Ca2+-